Gram negative

Ther Apher, 2002 Jun, 6(3), 221 - 4
A patient with severe acute pancreatitis successfully treated with a new critical care procedure; Moriguchi T et al.; It has been accepted widely that excessive humoral mediators play important roles in the pathogenesis of organ failure in patients with severe acute pancreatitis (SAP) and that infection of the pancreas due to bacterial translocation (BT) is the most frequent cause of death in SAP . On the other hand, it has been reported that continuous hemodiafiltration (CHDF) removes humoral mediators on hypercytokinemic patients such as those with systemic inflammatory response syndrome . Furthermore, several clinical studies have demonstrated that selective digestive decontamination (SDD) effectively eliminates aerobic Gram-negative bacteria from the intestinal tract and reduces the incidence of septic complications in SAP . Herein we report a case of SAP who was treated successfully with intensive care including CHDF and SDD . Thus, this case report suggests that CHDF aimed at removing causative humoral mediators and SDD for the prevention of BT are useful new tools for the management of SAP.

Can J Microbiol, 2002 May, 48(5), 458 - 62
Effect of fur mutation on acid-tolerance response and in vivo virulence of avian septicemic Escherichia coli; Zhu C et al.; The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria . In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined . The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain . However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation . These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E . coli, as detected by the ability to cause septicemia in our experimental infection.

Przegl Lek, 2002, 59 Suppl 1, 95 - 9
{Prospective study of prevalence hearing loss in preterm neonates in an intensive care unit}; Tomasik T; INTRODUCTION: The risk of hearing loss is higher in prematurely born infants compared with neonates born at term . It is due to unfinished development of auditory path and exposition to many harmful factors related to treatment in a neonatal intensive care unit . THE AIM: Assessment of hearing loss risk factors in preterm babies . PATIENTS: 152 newborns with mean birth weight 1408 +/- 551 g and gestational age 30.3 +/- 3.2 weeks . METHODS: After discharge follow-up (Me = 32 months) study of newborns born before 37 weeks of gestation age . The patients were assigned into two groups: A) with hearing loss (n = 9) and B) without hearing loss (n = 143) according to the results of clinical observation and audiological assessment after 6 months of life . RESULTS: The rate of hearing loss was higher in new-borns born between 24-28 weeks of gestation (6/50, 12%) than in those born between 29-32 (2/52.3%) and 33-36 (1/43, 2%, p = 0.03) . Identified risk factors independent of gestational age were: Gram negative sepsis (OR = 6; 95%CI: 1.32-27.33) and hyperbilirubinemia > 340 mumol/l (OR = 40.5; 95%CI: 73.27-503) . The other risk factors are: shock (OR = 6.65; 95%CI: 1.44-30.64), hypercarbia (pCO2 > 80 mmHg) (OR = 5.13; 95%CI: 1.29-20.37), severe anemia (Ht < 24%) (OR = 6.0; 95%CI: 1.32-27.33) . Prolonged treatment with aminoglicosides (over 10 days) also increased hearing loss risk (OR = 10.4; 95%CI: 1.27-85.75) . CONCLUSIONS: The risk of hearing loss is highest in the newborn born between 24-28 weeks of gestation . It can be further increased by shock, hypercapnia, severe anemia, prolonged treatment with aminoglicosides . Gestational age independent factors are: hyperbilirubinemia and Gram negative sepsis.

Przegl Lek, 2002, 59 Suppl 1, 63 - 6
{Influence of the lactose free and lactose containing diet on prevalence of gram-negative sepsis and feeding intolerance in very low birth weight infants: double-blind randomized trial}; Kwinta P et al.; OBJECTIVE: VLBW infants have a developmental lactase deficiency in the gut . The aim of the study was to evaluate the influence of lactose containing and lactose free diets on prevalence of feeding intolerance and Gram negative sepsis in VLBW infants . METHODS: 80 newborns with mean (+/- SEM) birth weight 1091 +/- 25 g and gestational age 28.5 +/- 0.24 wks were randomized into 2 groups fed during 1st month of life with 1) formula containing lactose (Bebilon Nenatal--BN group; n = 40) or 2) lactose free formula (Pregestimil--PG group; n = 40) . The end points of the study were: feeding intolerance episodes, Gram negative sepsis, weight gain and the length of parenteral nutrition . RESULTS: The birthweight (1112 vs 1114 g), gestational age (28.8 vs 28.3 wks), 5th min . Apgar score (5 vs 6 pts), sex (52% vs 55% male), type of delivery (65 vs 58% vaginal delivery) did not differ between the groups . Thirty (75%) newborns of the BN group and 31 (77.5%) newborns of the PG group completed the study (RR = 1.11; 95% CI: 0.54-2.44) . The prevalence of Gram negative sepsis were similar in both groups (2/40 vs . 3/40; RR = 0.67; 95% CI: 0.12-3.78) . Also a comparable number of children had at least one episode of feeding intolerance (14/40 vs . 12/40; RR = 1.17; 95% CI: 0.69-2.20) The weight gain (11.7 vs 10.9 g/day) and the length of parenteral nutrition did not differ between the groups (16 vs 15 days) . CONCLUSION: The inclusion of lactose into feeding formula does not influence feeding tolerance in VLBW infants.

J Bacteriol, 2002 Aug, 184(15), 4233 - 9
Role of the murein precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala in repression of beta-lactamase induction in cell division mutants; Uehara T et al.; Certain beta-lactam antibiotics induce the chromosomal ampC beta-lactamase of many gram-negative bacteria . The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway . Surprisingly, it has been reported that beta-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation . Since these results remain unexplained, we examined an ftsZ mutant and other cell division mutants (ftsA, ftsQ, and ftsI) and a PBP2 mutant for induction of beta-lactamase . In all mutants, beta-lactamase was not induced by cefoxitin, which confirms the initial reports . The murein precursor, UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress beta-lactamase expression in vitro . Our results suggest that beta-lactamase is not induced because the fts mutants contain a greatly increased amount of corepressor which the inducer cannot displace . In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available . Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.

Am J Transplant, 2001 Nov, 1(4), 366 - 72
Split liver transplantation for two adult recipients: an initial experience; Humar A et al.; The shortage of cadaver donor livers has been most severe for adult patients . Split liver transplantation is one method to expand the donor pool, but to have a significant impact on the waiting list, it needs to be applied for 2 adult recipients . We split livers from 6 cadaver donors, and transplanted 12 adult recipients . All splits were performed in situ with transection through the midplane of the liver, resulting in a right lobe and a left lobe graft . Mean donor age was 19.7 years; mean donor weight was 79.1 kg . Mean recipient age was 41.5 years . Mean weight of right lobe recipients was 89 kg; left lobe recipients, 60 kg . All donors were hemodynamically stable and had normal liver function tests . Mean operative time for the procurement was 7.4 h . Average blood loss during the transection of the liver was 490 mL . Mean GW/ RW ratio for all recipients was 0.87%; right lobe recipients, 0.86%; and left lobe recipients, 0.88% . With mean follow-up of 9.3 months, patient and graft survival rates were both 83.3% . There were 2 deaths: 1 after hepatic artery thrombosis (HAT) and subsequent multiorgan failure; the other after HAT, a liver retransplant, and subsequent gram-negative sepsis . The remaining 10 recipients are doing well . We observed no cases of primary nonfunction . Other complications included bile leak and/or stenosis (n = 3), bleeding from the Roux loop (n = 1), bleeding after percutaneous biopsy (n = 1), and incisional hernia (n = 1) . In conclusion, split liver transplantation, using 1 cadaver liver for 2 adult recipients, can be performed successfully . Crucial to success is proper donor and recipient selection.

Rev Gastroenterol Peru, 2002 Apr-Jun, 22(2), 159 - 63
{Treatment of gastritis-associated Helicobacter pylori infection in children}; Velasco Benitez CA et al.; Helicobacter pylori (Hp) the gram-negative bacteria succeeding in surviving in such an unreceptive medium as the gastric mucosa, has been associated with an important group of diseases in childhood with subsequent consequences in adulthood.Evidence has shown that Hp optimum treatment requires multi-drug regime due to its high ability to become resistant . This article presents the most recommended courses of therapy to treat Hp, as well as re-infection and resistance rates worldwide, analyzing the existing policies for Hp eradication and follow-up . Finally, we include our personal experience in prevalence and management of Hp found in endoscopies.

J Biol Chem, 2002 Sep 6, 277(36), 33378 - 85 Epub 2002 Jul 03.
Characterization of recombinant soluble macrophage scavenger receptor MARCO; Sankala M et al.; MARCO is a type II transmembrane protein of the class A scavenger receptor family . It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain . Previous studies have indicated a role for this receptor in anti-microbial host defense functions . In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties . The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system . Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure . Rotary shadowing also demonstrated that the molecules often associate with each other via the globes . sMARCO was found to bind avidly both heat-killed and living bacteria . Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO . Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition . Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied . Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity . Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.

Shock, 2002 Jul, 18(1), 69 - 74
Differences in neutrophil death among beta-lactam antibiotics after in vitro killing of bacteria; Matsuda T et al.; Antibiotic therapy is an essential treatment for gram-negative bacterial infections . Antibiotic-induced endotoxin release and subsequent production of inflammatory cytokines reportedly depend on the type of antibiotic action . This study examined the effects of various beta-lactam antibiotics on cell death of human polymorphonuclear neutrophils (PMNs) cocultured with Escherichia coli (E . coli) in vitro . E . coli morphology after antibiotic treatment was determined . PMNs and E . coli were cocultured with antibiotics for 0, 4, or 12 h . Levels of endotoxin and cytokines (TNF-alpha, IL-1beta, and IL-6) in the supernatants were measured . The filtrates of antibiotic-treated E . coli supernatants were cocultured with PMNs for 0, 4, or 12 h . In all experiments, ampicillin (ABPC), cefazolin sodium (CEZ), cefoperazone sodium (CPZ), latamoxef sodium (LMOX), imipenem (IPM), and polymyxin B sulfate (PLB) were used at 30 microg/mL . PMNs were isolated from healthy volunteers . PMN cell death was assessed by flow cytometry and light microscopy . ABPC, CEZ, CPZ, and LMOX, which induce bacterial filament formation with lysis, caused PMN necrosis when cocultured with E . coli . In contrast, IPM, which induces bacterial spheroplast formation with lysis, caused PMN apoptosis . Levels of endotoxin, TNF-alpha and IL-6 in the supernatants with IPM and PLB were significantly lower than in those with other beta-lactam antibiotics . The filtrates of IPM- and PLB-treated E . coli supernatants induced PMN apoptosis, whereas those treated with other beta-lactam antibiotics increased PMN necrosis . Beta-lactam antibiotics have different impacts on the types of PMN cell death after E . coli killing . Underlying mechanisms and the clinical relevance of IPM-induced PMN apoptosis in severe gram-negative infection warrant further investigation.

Folia Microbiol (Praha), 2002, 47(3), 235 - 40
Oxidative stress-induced expression of catalases in Comamonas terrigena; Zamocky M et al.; When grown under oxidative stress, catalatic as well as peroxidatic activity is increased in the Gram-negative bacterium Comamonas terrigena N3H . Two distinct hydroperoxidases were demonstrated by a specific staining . Based on their molar masses and their sensitivity toward 3-amino-1,2,4-triazole and high temperatures, they were identified as dimeric catalase-1 (Cat-1; 150 kDa), and as a tetrameric catalase-2 (Cat-2; 240 kDa) with enhanced peroxidatic activity, respectively . These two catalases differ in their expression during the bacterial growth; whereas the expression of the smaller enzyme (Cat-1) is induced by 0.5 mmol/L peroxides in the medium, and to a lesser degree by 25 mg/L Cd2+, Cat-2 (typical catalase) is almost specifically induced with cadmium ions.

Nat Biotechnol, 2002 Aug, 20(8), 839 - 42 Epub 2002 Jul 01.
Genome-wide internal tagging of bacterial exported proteins; Bailey J et al.; As a result of the explosive growth of bacterial genomic and postgenomic information, there is a pressing need for efficient, inexpensive strategies for characterizing the in vivo behavior and function of newly identified gene products . We describe here an internal tagging procedure, based on transposon technology, to facilitate the analysis of membrane-bound and secreted proteins in Gram-negative bacteria . The technique is based on a broad host range transposon (ISphoA/hah), which may be used to generate both alkaline phosphatase (AP) gene fusions and 63-codon in-frame insertions in the genome . The 63-codon insertion encodes an influenza hemagglutinin epitope and a hexahistidine sequence, permitting sensitive detection and metal affinity purification of tagged proteins . For each gene targeted, it is thus possible to monitor the disruption of phenotype (using the transposon insertion), the gene's transcription and translation (using the AP reporter activity), and the behavior of the unfused protein (using the internal tag) . Studies on a sequence-defined collection of Escherichia coli strains generated using the transposon showed that the synthesis and subcellular localization of tagged proteins could be readily monitored . The use of ISphoA/hah should provide a cost-effective approach for genome-wide in vivo studies of the behavior of exported proteins in a number of bacterial species.

Int Dent J, 2002 Jun, 52 Suppl 3, 229 - 32
Effect of deglycosylation of salivary glycoproteins on oral malodour production; Sterer N et al.; Putrefaction of saliva is commonly used as an in-vitro assay in oral malodour investigations . AIM: To exam the hypothesis that deglycosylation of salivary glycoproteins promotes oral malodour production . DESIGN: Porphyromonas gingivalis-mediated putrefaction of salivary glycoproteins was tested following preincubation of saliva in the presence of beta-galactosidase with or without glycosidic inhibitor (galactosamine), and in the presence of glucose with or without a non-glycosylated protein (bovine serum albumin) . METHODS: Malodour was determined by two odour judges, and volatile sulphides by using a sulphide monitor . Salivary glycoprotein degradation was measured densitometrically following electrophoresis on SDS-PAGE . RESULTS: The addition of beta-galactosidase promoted salivary glycoprotein degradation and concomitant malodour production, whereas addition of a glycosidic inhibitor (D-galactosamine) inhibited this process . Glucose inhibited salivary glycoproteins putrefaction, but this inhibitory effect was mitigated when a non-glycosylated protein (BSA) was added . CONCLUSIONS: Deglycosylation of salivary glycoproteins may be an initial step in oral malodour production . This process exposes the protein core of the glycoprotein, which is then further degraded by Gram-negative microorganisms under anaerobic conditions.

Int Dent J, 2002 Jun, 52 Suppl 3, 221 - 8
Cysteine challenge testing: a powerful tool for examining oral malodour processes and treatments in vivo; Kleinberg I et al.; Gram-negative oral bacteria rapidly produce the odorant hydrogen sulphide from cysteine . It provides a major part of the oral malodour bouquet while causing a corresponding decrease in the oxidation-reduction potential (Eh) . A low Eh favours oral putrefaction and malodour occurrence . Challenge testing with cysteine (5ml of 6mM for 30 seconds) enabled evaluation of: the contribution of tongue and teeth bacteria to overall oral malodour; the effectiveness of tongue and tooth brushing, tooth scraping, gum chewing and mouthrinsing with different agents in reducing oral malodour . Successive cysteine challenge tests for 20 minute periods at selected times in a seven hour experiment were effective for assessing the magnitude and duration of an agent's effectiveness . Brushing the teeth reduced malodour modestly . So did tongue scraping and gum chewing . In contrast, brushing the tongue dorsum, especially the posterior half was remarkably effective, which confirmed it as a major site of oral malodour contribution . Rinses containing various actives showed wide variation in effectiveness . The experiments demonstrated that cysteine challenge testing is potentially a aluable tool for assessing the ability of the oral bacteria to produce malodour and for assessing agents designed to inhibit such production.

J Clin Microbiol, 2002 Jul, 40(7), 2693 - 5
Bacteremia due to Moraxella atlantae in a cancer patient; De Baere T et al.; A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient . No identification could be reached using phenotypic techniques . Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library . Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection . In retrospect, the phenotypic characteristics fit the identification as M . atlantae (formerly known as CDC group M-3) . Comparative 16S rRNA sequence analysis indicates that M . atlantae, M . lincolnii, and M . osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.

Paediatr Drugs, 2002, 4(7), 469 - 84
Efficacy and tolerability of extended-interval aminoglycoside administration in pediatric patients; Kraus DM et al.; Aminoglycosides are commonly used to treat serious Gram-negative infections in pediatric patients . An effort to improve the efficacy and tolerability of this antibiotic class has led to evaluation of extended-interval aminoglycoside administration (EIAA) . EIAA is designed to achieve higher peak plasma aminoglycoside concentrations, with relatively undetectable trough concentrations, when compared with conventional aminoglycoside administration (CAA), and is therefore expected to be markedly effective and to reduce drug accumulation and prevent nephrotoxicity and ototoxicity . Clinical trials evaluating EIAA in neonates included patients with suspected Gram-negative infections requiring short courses of aminoglycoside therapy . Consequently, comparative efficacy of EIAA versus CAA could not be assessed . In addition, ototoxicity was often not assessed, and nephrotoxicity was virtually undetectable . Similarly, trials evaluating EIAA versus CAA in infants and children have not demonstrated a difference in outcomes . The use of EIAA in children with febrile neutropenia has been evaluated primarily with amikacin . The incidences of nephrotoxicity and ototoxicity were low, and were similar between EIAA and CAA . No deaths were reported in any of these studies; however, this could be related to the inclusion of patients with undocumented bacteremia . Further investigation of EIAA is necessary in patients with documented bacteremia, since plasma aminoglycoside concentrations were undetectable for most of the dosage interval in children with febrile neutropenia who were treated once daily . Overall, clinical studies suggest that EIAA has similar efficacy to, and no higher risk of toxicity than, CAA in neonates, infants, and children . A few evaluations have also demonstrated that EIAA is cost-effective in neonates and in children with febrile neutropenia . Future studies evaluating the efficacy and tolerability of EIAA in pediatric patients with documented systemic infections should be prospective, randomized, controlled trials with sample sizes sufficient to detect differences between administration methods . Further evaluations should also address the optimal dosage and cost-effectiveness of EIAA in infants and children.

J Bacteriol, 2002 Jul, 184(14), 3808 - 14
The RNA polymerase alpha subunit from Sinorhizobium meliloti can assemble with RNA polymerase subunits from Escherichia coli and function in basal and activated transcription both in vivo and in vitro; Peck MC et al.; Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family . To facilitate our studies of transcription in S . meliloti, we cloned and characterized the gene for the alpha subunit of RNA polymerase (RNAP) . S . meliloti rpoA encodes a 336-amino-acid, 37-kDa protein . Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (alpha)-rplQ (L17) found in the alpha-proteobacteria . In vivo, S . meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E . coli rpoA, demonstrating that S . meliloti alpha supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E . coli . In vitro, we reconstituted RNAP holoenzyme from S . meliloti alpha and E . coli beta, beta', and sigma subunits . Similar to E . coli RNAP, the hybrid RNAP supported transcription from an E . coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation . We obtained similar results using purified RNAP from S . meliloti . Our results demonstrate that S . meliloti alpha functions are conserved in heterologous host E . coli even though the two alpha subunits are only 51% identical . The ability to utilize E . coli as a heterologous system in which to study the regulation of S . meliloti genes could provide an important tool for our understanding and manipulation of these processes.

J Mol Biol, 2002 Jun 28, 320(1), 23 - 37
Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF; Elderkin S et al.; The PspA protein, a negative regulator of the Escherichia coli phage shock psp operon, is produced when virulence factors are exported through secretins in many Gram-negative pathogenic bacteria and its homologue in plants, VIPP1, plays a critical role in thylakoid biogenesis, essential for photosynthesis . Activation of transcription by the enhancer-dependent bacterial sigma(54) containing RNA polymerase occurs through ATP hydrolysis-driven protein conformational changes enabled by activator proteins that belong to the large AAA(+) mechanochemical protein family . We show that PspA directly and specifically acts upon and binds to the AAA(+) domain of the PspF transcription activator . Interactions involving PspF and nucleotide are changed by the action of PspA . These changes and the complexes that form between PspF and PspA can explain how PspA exerts its negative effects upon transcription activated by PspF, and are of significance when considering how activities of other AAA(+) proteins might be controlled . (c) 2002 Elsevier Science Ltd.

Rinsho Byori, 2002 May, 50(5), 519 - 23
{Plasma endotoxin assay by supersensitive reagent for the diagnosis of gram-negative bacteremia}; Tobita T et al.; With a novel supersensitive reagent, we evaluated the utility of measuring plasma endotoxin level for the rapid and sensitive diagnosis of gram-negative bacteremia . Subjects were 112 febrile(more than 38 degrees C) patients suspected of having bacterial infection and 170 samples were collected . Venous blood was obtained aseptically before administering antibiotics . Blood culture and endotoxin assays were performed simultaneously with these materials . Plasma endotoxin levels were positive in 64 samples when the cut off index was postulated at 0.35 pg/ml, while only 5 samples when the cut off index was postulated at 5 pg/ml . When the cut off index was at 0.35 pg/ml, sensitivity was 86%, while it was 14% when the cut off index was 5 pg/ml . Gram-negative rods(GNR) were detected by blood culture in 14 cases and the average period to detect GNR was 14.8 hours . The presence of circulating viable bacteria is diagnosed by blood culture, but because of the serious consequence of bacterial sepsis, treatment is initiated even in the absence of an identifiable organism . Since plasma endotoxin level can be assayed in about 2 hours, it will be more practical if we adjust the cut off index according to the clinical situation.

Crit Care Med, 2002 Jun, 30(6), 1327 - 33
Selective decontamination of the digestive tract: impact on cytokine release and mucosal damage after hemorrhagic shock; Kahlke V et al.; OBJECTIVE: To determine if selective decontamination of the digestive tract (SDD) influences the proinflammatory immune response of the gut after hemorrhage . DESIGN: Random assignment to either unmanipulated control after 7 days of SDD or conventional rat chow, or hemorrhagic shock group after the same time of conventional rat chow or SDD . SETTING: University animal laboratory . SUBJECTS: Male Wistar rats, weighing between 300 and 350 g . INTERVENTION: Animals of the control group were not manipulated until organ harvesting, whereas animals of the hemorrhagic shock group were bled to 30 +/- 5 mm Hg for 90 mins by withdrawal/reinfusion of shed blood and were resuscitated by Ringer's lactate equivalent to the shed blood volume . MEASUREMENTS AND MAIN RESULTS: Rats were killed after resuscitation (hemorrhagic shock group) or completed feeding (control group) . Whole portal and caval blood was obtained, and splenic macrophages and gut mononuclear cells were harvested to measure supernatant tumor necrosis factor-alpha and IL-6 by bioassay . Mesenteric lymph nodes were obtained to determine bacterial translocation, and a histologic specimen was taken from the distal ileum . Feces were harvested to examine the effect of SDD . SDD eliminated Gram-negative enteric bacteria and had no influence on mucosal damage or on bacterial translocation in control animals and animals after hemorrhage . In animals receiving conventional rat chow, hemorrhagic shock led to significantly (p <.05) elevated lipopolysaccharide-stimulated proinflammatory cytokine (tumor necrosis factor-alpha and interleukin-6) release in whole portal blood, splenic macrophages, and gut mononuclear cells compared with the control group without shock . In contrast, hemorrhagic shock after SDD led to suppressed or unchanged cytokine release compared with unmanipulated animals receiving SDD . However, SDD itself induced significant (p <.05) cytokine release in these organs . Furthermore, plasma concentrations of tumor necrosis factor-alpha and interleukin-6 were significantly (p <.05) elevated in animals after hemorrhage and SDD compared with animals after hemorrhage alone . CONCLUSIONS: Hemorrhagic shock led to significant cytokine release . In contrast, cytokine release after hemorrhage and SDD was unchanged or suppressed . Furthermore, in control animals without hemorrhagic shock, SDD induced significant cytokine release . Therefore, selective decontamination of the gut, as practiced in some patients, may induce additional proinflammatory cytokine release, which can add to the proinflammatory burst in case of a complication such as hemorrhagic shock.

Protein Expr Purif, 2002 Jun, 25(1), 134 - 7
A rapid selective extraction procedure for the outer membrane protein (OmpF) from Escherichia coli; Arcidiacono S et al.; Porins are essential pore-forming proteins found in the outer membrane of several gram-negative bacteria . Investigating the relationships between molecular structure and function involves an extremely time-consuming and labor-intensive purification procedure . We report a method for rapid extraction of the outer membrane protein, OmpF, from freeze-dried Escherichia coli cells using valeric acid, alleviating the effort and time in sample preparation . Extraction results in a highly enriched fraction containing OmpF as 76% of the total protein content . The apparent molecular mass determined by SDS-PAGE mobility was 38,900, similar to that of the monomeric form of OmpF . N-terminal sequencing yielded 23 amino acids with 100% identity to the published OmpF sequence . The trimeric form of OmpF was observed in unheated samples run on SDS-PAGE and analysis of these samples by periodic acid/silver staining revealed the presence of unbound lipopolysaccharides . Furthermore, this method should prove useful for isolating other outer membrane proteins .

Med Sci Monit, 2002 Jun, 8(6), HY15 - 8
Modulation of the susceptibility of intestinal bacteria to bacteriophages in response to Ag43 phase variation -- a hypothesis; Wegrzyn G et al.; Escherichia coli is a Gram-negative bacterium which colonizes the intestinal tract of man and other animals . In addition to being a part of the normal bacterial flora of the human intestine, there are a number of enteropathogenic strains of E . coli which cause infections ranging in consequence from diarrhoea to colitis . Antigen 43 (Ag43) is the major phase-variable protein in the outer membrane of E . coli . One benefit for bacteria resulting from phase variation of surface antigens is usually ascribed to evasion of host defences . However, results of recent studies indicate that infection of E . coli by different bacteriophages is inhibited in the presence of certain bile salts and carbohydrates (components present in the human intestine but absent in standard bacteriological media) when cells are in the 'OFF' state for production of Ag43 . The inhibition of bacteriophage development was found to be due to a significant impairment in the process of phage adsorption and evidence was presented for the binding of phage to Ag43 . Here we present a hypothesis that in the case of Ag43, phase-variation might benefit the host bacterium by modulating the susceptibility to phage infection in the gut . If this hypothesis is true, it may have important implications not only for basic research but also for development of bacteriophage therapy, a re-discovered method of treatment of patients with infectious diseases.

Shock, 2002 Jun, 17(6), 485 - 90
Globin attenuates the innate immune response to endotoxin; Yang H et al.; Hemoglobin is an endotoxin (lipopolysaccharide; LPS)-binding protein that synergistically increases the release of proinflammatory cytokines from the innate immune system in response to LPS . It has been suggested that this activity of hemoglobin facilitates the recognition of Gram-negative bacteria in a wound, thereby maximizing immune efficiency . This synergy may be important to the pathogenesis of a broad spectrum of clinical conditions because elevated hemoglobin levels frequently are observed in patients after the transfusion of red cells, trauma, cardiopulmonary bypass surgery, hemolysis, in addition to other disorders . To determine the molecular basis of the specific hemoglobin-LPS synergy, in this article we tested the effects of globin itself on macrophage responses to LPS . Paradoxically, these studies revealed that globin suppressed tumor necrosis factor (TNF) synthesis in LPS-stimulated murine and human macrophage cultures . LPS comigrated with globin on non-denaturing electrophoretic gels, giving direct evidence for binding . Globin specifically inhibited LPS activity in the standard Limulus assay but did not inhibit interleukin-1beta-mediated TNF synthesis . Iron supplementation of macrophage cultures significantly increased interleukin-1beta-induced TNF release . Intraperitoneal administration of globin protected mice against both LPS-induced lethality and experimentally induced bacterial infection . Thus, the heme-iron moiety of hemoglobin, and not the binding of LPS to globin, enhanced macrophage responses to LPS.

Mol Microbiol, 2002 Jun, 44(5), 1131 - 9
An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations; Andersen C et al.; The TolC protein of Escherichia coli comprises an outer membrane beta-barrel channel and a contiguous alpha-helical tunnel spanning the periplasm, providing an exit duct for protein export and multidrug efflux . It forms a single transmembrane pore that is open to the outside of the cell but constricted at the peri-plasmic tunnel entrance . This sole constriction is lined by a ring of six aspartate residues, two in each of the three identical monomers . When these were replaced by alanines, the resulting TolC(DADA) protein reconstituted normally in black lipid membranes but showed altered electrophysiological characteristics . In particular, it had lost the strong pH dependence of the wild type and had switched ion selectivity from cations to anions . The function of wild-type TolC as a membrane pore was severely inhibited by divalent and trivalent cations entering the channel tunnel from the channel ("extracurricular") side . Divalent cations bound reversibly to effect complete blocking of the transmembrane ion flux . Trivalent cations were more potent . Hexamminecobalt bound at nanomolar concentrations allowed visualization of single blocking events, whereas the smaller Cr(3+) cation bound irreversibly and could also access the cation binding site via the tunnel entrance . The inhibitory cations had no effect on the mutant TolC(DADA), supporting the view that the aspartate ring is the cation binding site . The electronegative entrance is widely conserved throughout the TolC family, which is essential for efflux and export my Gram-negative bacteria, suggesting that it could present a general target for drugs.

Dis Colon Rectum, 2002 Mar, 45(3), 394 - 400
A low-morbidity murine model of peritonitis; Cooper D et al.; PURPOSE: Peritonitis continues to be a major source of mortality and morbidity in patients undergoing abdominal surgery . The aim of this study was to develop a nonfatal model of bacterial peritonitis in mice so that we could study aspects of the pathobiology and treatment of peritonitis in an in vivo model . METHODS: Mice were inoculated via a midline laparotomy with 0.5 mg of zymosan in 0.1 ml of saline into the subomental space . At 24, 48, and 96 hours after treatment, animals were killed, and analysis was performed to determine the degree of peritoneal inflammation . End points included intraperitoneal cellular influx, tumor necrosis factor-alpha concentrations, and myeloperoxidase activity . In addition, peritoneal lavage fluid was plated onto blood agar for analysis of bacterial colony-forming units . There were 40 mice in each group . RESULTS: There were no deaths in either group . Facultative Gram-negative bacteria were cultured from the peritoneal cavities of zymosan-treated animals at 24 and 48 hours after insult (colony-forming unit counts of 92+/-11 vs . 0 in control animals) . In the zymosan-treated animals, there were significantly increased numbers of inflammatory cells (especially neutrophils) in the peritoneal cavity at 24 and 48 hours after treatment; these numbers returned to control levels by 96 hours . Myeloperoxidase activity was also elevated both in the peritoneal fluid (2.4 X 10(-4) units/ml compared with 1.6 x 10(-4) units/ml, P < 0.05) and in remote organs (i.e., lung, P < 0.05; liver, P < 0.001; and kidney, P < 0.05) at 24 hours after treatment . There was no significant difference in tumor necrosis factor-alpha levels between zymosan-treated and control mice in either serum or peritoneal fluid at any time point investigated . There was no mortality in either the zymosan-treated or control animals . CONCLUSION: In this model of bacterial peritonitis in mice, we have demonstrated how the peritoneal cavity can resolve a relatively localized inflammatory insult within 96 hours of induction . This response is characterized by a cellular influx of predominantly neutrophils and macrophages and by pronounced oxidative activity . We will use this in vivo model to characterize aspects of the pathobiology and treatment of peritonitis.

Avian Dis, 2002 Apr-Jun, 46(2), 453 - 60
Seroprevalence of antibodies specific for gram-negative core antigens in chickens on the basis of an Escherichia coli J5 enzyme-linked immunosorbent assay; Ruble RP et al.; Antibodies directed toward gram-negative core antigens (GNCAs) have been demonstrated in many mammalian species but to date are unexamined in any avian species . An enzyme-linked immunosorbent assay with phenol-killed whole cell Escherichia coli J5 was used to assess the presence of serum antibodies directed toward GNCAs in chickens . The first experiment consisted of collecting blood samples from randomly selected hens at egg laying ranches in northern California . The ages ranged from several days of age to 77 wk of age . Birds were classified into age groups (hatchling {1 day-4 wk}, pullet {4-18 wk}, pullet cycle {18-60 wk}, and postmolt {>60 wk}) and husbandry style for titer comparison . The geometric mean titer (GMT) for all adult hens regardless of age was 2147 . The geometric mean titers were 220, 5691, 2304, and 1776 for hatchlings, pullets, pullet cycle hens, and postmolt hens, respectively . The age group titer trends were similar to those of humans rather than those of farm animals in that the highest titers occurred during "adolescence" (pullets) and titers decreased slightly with maturity . The GMTs were 2870 for hens housed intensively and 1872 for those housed extensively . The second experiment looked at the progression of GNCA titers within individual birds over a 1-yr period . Individual titers increased slightly throughout the study time of the second experiment.

Avian Dis, 2002 Apr-Jun, 46(2), 447 - 52
Isolation of an unidentified, nonfermentative, gram-negative bacterium from turkeys and chickens: 38 cases (1995-2001); Chin RP; Thirty-eight cases were identified in which a nonfermentative, gram-negative, rod-shaped bacterium was isolated from the respiratory system of turkeys and chickens . Cases were submitted from various parts of the country . Preliminary assessment of phenotypic characteristics indicated this bacterium was different from common pathogenic or opportunistic bacteria isolated from the avian respiratory tract . Most cases reported a history of respiratory distress and/or increased flock mortality . Lesions seen in infected birds included tracheitis and pneumonia, which correlate with the sites of isolation . Sixty-one percent of the isolations were made from the trachea and 25% from the lung . Age of infected birds ranged from 35 to 315 days in turkeys and 53 days to 3 yr in chickens . In most instances (90%), other bacteria were also isolated from affected sites . The significance of this organism in respiratory disease in birds is unknown.

Cas Lek Cesk, 2002 May 10, 141(9), 270 - 5
{Tularemia--history, epidemiology, clinical aspects, diagnosis and therapy}; Cerny Z; Tularemia was first described 90 years ago by McCoy as a disease of animals . At the beginning of twenties it was recognised by E . Francis as a disease transmittable from animals to man . Tularemia is caused by a gram-negative microbe Francisella tularensis . Epidemiological and clinical manifestations of the disease are highly diverse . The characteristic sign is the primary complex consisting from an initial ulceration and a regional lymphadenitis . In the Czech republic tularemia was first identified in 1936 in the south of Moravia and for the next years it occurred sporadically or in epidemic form also in the western Moravia, in north-west and east of Bohemia . It affected persons manipulating with the diseased animals, namely with hares, workers in animal farms and those working in cold sections of sugar mills . After a longer pause, during the last six years, the incidence of tularemia has increased again . That is why we decided to renew the understanding of the disease.

Biochem Biophys Res Commun, 2002 Jun 14, 294(3), 560 - 6
Identification of a novel Comamonas testosteroni gene encoding a steroid-inducible extradiol dioxygenase; Skowasch D et al.; Comamonas testosteroni is a Gram-negative bacterium that can grow on steroids and polycyclic aromatic hydrocarbons (PAH) as sole carbon and energy source . Complete mineralisation of these compounds is achieved through complex metabolic pathways comprising a set of inducible enzymes . Whereas the degradation pathways for PAHs have been intensively studied, patterns of enzymes leading to ring fissions of the steroid nucleus are unclear . Several intermediates of the steroid and PAH degradation pathways have similar structures therefore the question remains of whether both classes are substrates of different degradation routes or whether some catabolic enzymes function in both pathways . Interestingly, our studies reveal that testosterone simultaneously induces the expression of steroid- and PAH-catabolising enzymes in C . testosteroni . By cloning the gene, one of these testosterone-inducible proteins (TIP1) turned out to be biphenyl-2,3-diol-1,2-dioxygenase . This enzyme has been described to convert 2,3-dihydroxybiphenyl into 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid in PAH degradation . The gene was found on a cluster encoding TIP1, three orfs, and another testosterone-inducible protein (TIP6) of unknown function . The deduced amino acid sequence of TIP1 revealed that the enzyme contains 299 amino acids (34 kDa) and shares homologies to a variety of other extradiol dioxygenases . Based on the similar catechol moieties in PAH and steroid intermediates, together with its inducibility by testosterone, it is conceivable that TIP1 functions as a steroid extradiol dioxygenase to convert steroidal secocatechols into the disecoandrostanes . Our data suggest a role of the reported TIP1 protein in both the degradation pathways for steroids and aromatic hydrocarbons.

J Biol Chem, 2002 Aug 30, 277(35), 32124 - 32 Epub 2002 Jun 06.
The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells; Guha M et al.; Monocytes and macrophages express cytokines and procoagulant molecules in various inflammatory diseases . In sepsis, lipopolysaccharide (LPS) from Gram-negative bacteria induces tumor necrosis factor-alpha (TNF-alpha) and tissue factor (TF) in monocytic cells via the activation of the transcription factors Egr-1, AP-1, and nuclear factor-kappa B . However, the signaling pathways that negatively regulate LPS-induced TNF-alpha and TF expression in monocytic cells are currently unknown . We report that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway enhances LPS-induced activation of the mitogen-activated protein kinase pathways (ERK1/2, p38, and JNK) and the downstream targets AP-1 and Egr-1 . In addition, inhibition of PI3K-Akt enhanced LPS-induced nuclear translocation of nuclear factor-kappa B and prevented Akt-dependent inactivation of glycogen synthase kinase-beta, which increased the transactivational activity of p65 . We propose that the activation of the PI3K-Akt pathway in human monocytes limits the LPS induction of TNF-alpha and TF expression . Our study provides new insight into the inhibitory mechanism by which the PI3K-Akt pathway ensures transient expression of these potent inflammatory mediators.

Vet Immunol Immunopathol, 2002 Aug, 87(1-2), 11 - 8
Lipopolysaccharide induces inflammatory cytokines in the pig amnion; Trebichavsky I et al.; Inflammatory mediators that are induced by gram-negative bacteria in the course of intrauterine infections threaten successful pregnancy . To compare the effect of two different routes of cytokine induction, bacterial lipopolysaccharide (LPS) was administered in vivo either into the cord vein or into the amniotic cavity of pig fetuses in the second half of gestation for 20 h and cytokines were detected in the amnion.Tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were induced in the amniotic epithelium after intra-amniotic but not after intra-venous administration of LPS . The presence of IL-8 was confirmed by RT-PCR . In contrast, transforming growth factor-beta1 (TGF-beta1) was expressed constitutively and was found in all samples of the amniotic epithelium.Amniotic fluid contained only minute levels of TNF-alpha . IL-8 levels in amniotic fluid increased after the treatment with LPS and the highest IL-8 levels were found in dead LPS-treated fetuses.

Arch Biochem Biophys, 2002 Jun 1, 402(1), 65 - 76
Isolation and characterization of an acetyl group-recognizing agglutinin from the serum of the Indian white shrimp Fenneropenaeus indicus; Maheswari R et al.; A natural agglutinin from the serum of the Indian white shrimp Fenneropenaeus (Penaeus) indicus was purified to electrophoretic homogeneity by a single-step affinity chromatography on N-acetylglucosamine-Sepharose 6B . The expression of hemagglutinating (HA) activity of F . indicus agglutinin (FIA) was independent of the presence of divalent cations and insensitive to their chelators . FIA gave a single symmetrical peak in its native form with a molecular mass estimate of 200 kDa on gel filtration in HPLC, and SDS-PAGE under reducing conditions revealed that it is a homo-oligomer of a 27-kDa subunit protein . The pattern of reactivity of FIA against anti-FIA rabbit serum in immunodiffusion and immunoelectrophoretic analysis provided additional evidence for its purity and homogeneity . HA-inhibition studies documented exclusive specificity of FIA for acetyl groups in carbohydrates independently of the presence of these groups at the C-2 or C-5 position and its stereochemical arrangement in the axial or equatorial orientation . The unique ability of FIA to recognize acetyl groups was also explicitly demonstrated with sialo- and asialo-glycoproteins . Strikingly, FIA also interacted equally with amino acids and chemicals containing acetyl groups, thereby unambiguously demonstrating the exquisite specificity of FIA for an acetyl group, irrespective of the presence of this group in carbohydrate or noncarbohydrate ligands . The susceptibility of HA activity of FIA to inhibition by lipopolysaccharides from diverse gram-negative bacteria as well as its ability to selectively agglutinate several bacterial species isolated from infected shrimps implicate a potential role of this humoral agglutinin of F . indicus in the host immunodefense reactions against microbial invaders.

Arch Biochem Biophys, 2002 Jun 15, 402(2), 159 - 65
Isomeric N-alkylpyridylporphyrins and their Zn(II) complexes: inactive as SOD mimics but powerful photosensitizers; Benov L et al.; The ortho, meta, and para isomers of cationic N-alkylpyridylporphyrins and their Zn(II) complexes were compared in terms of their photodynamic properties . The ortho Zn(II) complex was found to be the most efficient in causing photooxidation of NADH in vitro . In Escherichia coli, however, the para and meta isomers were better photosensitizers than their ortho analogs . The lower potency of the ortho compound in vivo seems to be due to its lower intracellular concentration . All porphyrins tested were more efficient in killing E . coli and in photooxidizing NADH than the hematoporphyrin derivative . Antibiotic resistance did not affect the photokill, which implies that the cationic N-alkylpyridylporphyrins, as their Zn(II) complexes, can be used as bactericidal agents against antibiotic-resistant strains of gram-negative bacteria.

Genome Biol . 2002;3(5):research0025 . Epub 2002 Apr 29.
Lateral gene transfer and parallel evolution in the history of glutathione biosynthesis genes; Copley SD et al.; BACKGROUND: Glutathione is found primarily in eukaryotes and in Gram-negative bacteria . It has been proposed that eukaryotes acquired the genes for glutathione biosynthesis from the alpha-proteobacterial progenitor of mitochondria . To evaluate this, we have used bioinformatics to analyze sequences of the biosynthetic enzymes gamma-glutamylcysteine ligase and glutathione synthetase . RESULTS: Gamma-glutamylcysteine ligase sequences fall into three groups: sequences primarily from gamma-proteobacteria; sequences from non-plant eukaryotes; and sequences primarily from alpha-proteobacteria and plants . Although pairwise sequence identities between groups are insignificant, conserved sequence motifs are found, suggesting that the proteins are distantly related . The data suggest numerous examples of lateral gene transfer, including a transfer from an alpha-proteobacterium to a plant . Glutathione synthetase sequences fall into two distinct groups: bacterial and eukaryotic . Proteins in both groups have a common structural fold, but the sequences are so divergent that it is uncertain whether these proteins are homologous or arose by convergent evolution . CONCLUSIONS: The evolutionary history of the glutathione biosynthesis genes is more complex than anticipated . Our analysis suggests that the two genes in the pathway were acquired independently . The gene for gamma-glutamylcysteine ligase most probably arose in cyanobacteria and was transferred to other bacteria, eukaryotes and at least one archaeon, although other scenarios cannot be ruled out . Because of high divergence in the sequences, the data neither support nor refute the hypothesis that the eukaryotic gene comes from a mitochondrial progenitor . After acquiring gamma-glutamylcysteine ligase, eukaryotes and most bacteria apparently recruited a protein with the ATP-grasp superfamily structural fold to catalyze synthesis of glutathione from gamma-glutamylcysteine and glycine . The eukaryotic glutathione synthetase did not evolve directly from the bacterial glutathione synthetase.

Biometals, 2002 Jun, 15(2), 133 - 44
New synthetic catecholate-type siderophores with triamine backbone; Heinisch L et al.; New analogues of triscatecholate siderophores based on linear or tripodal triamines with or without spacer groups or lipophilic and hydrophilic substituents were synthesized . The catecholate moieties were prepared in OH-forms, as acetylated compounds or masked as 8-methoxycarbonyloxy-2,4-dioxo-1,3-benzoxazine derivatives . Some of the new compounds were active as siderophores tested by growth promotion assays using various gram-negative bacteria and mycobacteria under iron limitation and by CAS-assay . Structure-activity-correlations have been studied.

Mol Microbiol, 2002 May, 44(4), 935 - 46
Genetic dissection of Ralstonia solanacearum hrp gene cluster reveals that the HrpV and HrpX proteins are required for Hrp pilus assembly; Van Gijsegem F et al.; In both plant and mammalian Gram-negative pathogenic bacteria, type III secretion systems (TTSSs) play a crucial role in interactions with the host . All these systems share conserved proteins (called Hrc in plant pathogens), but each bacterium also produces a variable number of additional type III proteins either unique or with counterparts only in a limited number of related systems . In order to investigate the role of the different proteins encoded by the hrp gene cluster of the phytopathogenic bacterium Ralstonia solanacearum, non-polar mutants in all hrp genes (except for hrcQ) were analysed for their interactions with plants, their ability to secrete the PopA protein and their production of the Hrp pilus . In addition to Hrc proteins and the HrpY major component of the Hrp pilus, four additional Hrp proteins are indispensable for type III secretion and for interactions with plants . We also provide evidence that hrpV and hrpX mutants can still target the HrpY pilin outside the bacterial cell but are impaired in the production of Hrp pili, indicating that HrpV and HrpX proteins are involved in the assembly of this appendage.

Annu Rev Biochem, 2002, 71, 635 - 700 Epub 2001 Nov 09.
Lipopolysaccharide endotoxins; Raetz CR et al.; Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating "core" oligosaccharide, and a distal polysaccharide (or O-antigen) . Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens . Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics . Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist . Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells . TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88 . The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 83 - 9
{Detection of the endotoxin of Gram-negative bacteria in human blood}; Bondarenko VM et al.; Recent new data on the important role played by lipopolysaccharides (endotoxin) of Gram negative bacteria in physiology and pathogenesis of the most important human infectious and noninfectious diseases testify to the necessity of wide clinical trials of different methods for LPS detection in blood and other physiological fluids . Among presently available diagnostic methods for endotoxinemia detection, the highly sensitive LAL (Limulus Amebocyte Lysate) test in various modifications is most widely used . The LAL test is known to be non-specific, however many drawbacks of this test have been successfully overcome . The results of clinical studies on the determination of the LPS activity in the systemic blood stream and antibody titers to its most common determinants, as well as the reserves of endotoxin binding with granulocytes give grounds for optimistic evaluation of the future studies on the role of LPS in human physiology and pathology . In clinical practice both positive sides and drawbacks of the presently known methods for LPS detection, including the LAL test, must be borne in mind for the complex evaluation of endotoxinemia levels.

J Chemother, 1991 Jan, 3 Suppl 1, 205 - 7
Imipenem-cilastatin in the treatment of hospital infections; Mao P et al.; Imipenem was tested on 65 gram-negative bacterial strains consecutively isolated in patients affected by hospital infection and used as empiric therapy in 52 patients presenting a hospital infection suspected of gram-negative origin . More than 96% of the tested strains resulted sensitive to imipenem . This antibiotic showed a good clinical result in more than 80% of the cases when used as therapy . Therefore imipenem could be considered the drug of first choice in patients with a severe prognosis and a nosocomial infection suspected to be due to a gram-negative microorganism.

J Chemother, 1991 Jan, 3 Suppl 1, 201 - 4
Effect of antibiotics on polycation-treated Escherichia coli HB101 (pRI203); Valenti P et al.; In the present paper, we demonstrated that low concentrations of various polycationic agents sensitize E . coli HB 101 harboring the plasmid pRI203, containing the Y . pseudotuberculosis invasion region, to antibiotics rifampicin, amikacin, ceftazidime and cefotaxime . These antibiotics, known to be excluded, to various degrees, by the bacterial outer membrane, resulted several-fold more active towards polycation-treated bacteria by comparison with controls . This increased permeability to antibiotics of E . coli HB 101 (pRI203) probably depends upon the binding of polycations to the acidic moieties of lipopolysaccharide, as already suggested for other gram-negative bacteria.

Clin Exp Immunol, 2002 May, 128(2), 221 - 8
Interleukin-1beta induces in vivo tolerance to lipopolysaccharide in mice; Alves-Rosa F et al.; Endotoxin or lipopolysaccharide (LPS) tolerance may be partially due to the secretion of potent anti-inflammatory cytokines following severe Gram-negative infections, or by low doses of LPS . In this work, we describe the effects of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF-), two early cytokines secreted after LPS exposure, in the induction of LPS tolerance . Our results demonstrate that mice treated with three daily doses of 100 ng of IL-1 were tolerant to LPS-induced shock . However, TNF- was unable to induce an LPS refractory state . Given the fact that 100 ng of IL-1 increase the plasma levels of glucocorticoids, we evaluated whether a daily injection of dexamethasone (DEX) alone was able to reproduce the LPS-like tolerant state . However, no signs of LPS refractoriness were detected, except when DEX was administered concomitantly with a dose of IL-1 that does not induce corticosterone secretion (12 ng/mouse) . This dose was found to induce in vitro up-regulation of the glucocorticoid receptors (GcR) of peritoneal macrophages following 24 h of treatment . In addition, we demonstrate that IL-1 is capable of inducing the down-regulation of Toll-like receptor 4 (TLR4), a crucial molecule in the signal transduction of LPS . Taken together, our results indicate that IL-1 can generate tolerance to LPS in vivo, and suggest that the regulation of mechanisms of the down-regulation of TLR4, as well as those involved in the expression of GcR and/or in the secretion of glucocorticoids, would be crucial for these effects.

Methods Find Exp Clin Pharmacol, 2002 Mar, 24(2), 67 - 70
Surface expression of toll-like receptor 4 on THP-1 cells is modulated by Bu-Zhong-Yi-Qi-Tang and Shi-Quan-Da-Bu-Tang; Mita Y et al.; Human Toll-like receptor 4 (TLR4) has recently been identified and has been shown to be the main protein involved in recognizing Gram-negative bacteria . We examined the regulation of TLR4 surface expression in a human monocytic cell line (THP-1 cells) by two traditional Chinese herbal medicines . Bu-Zhong-Yi-Qi-Tang (TJ-41) and Shi-Quan-Da-Bu-Tang (TJ-48) . TJ-41 and TJ-48 upregulated TLR4 surface expression in THP-1 cells, as well as enhanced TLR4 surface expression in these cells both dose- and time-dependently . These findings suggest that TJ-41 and TJ-48 increase the receptor involved in the response to Gram-negative bacteria and may enhance defenses against these pathogens.

J Clin Microbiol, 2002 Jun, 40(6), 1908 - 12
Captive rhesus monkeys (Macaca mulatta) are commonly infected with Helicobacter cinaedi; Fernandez KR et al.; Helicobacter cinaedi may cause proctocolitis or bacteremia in homosexual men infected with human immunodeficiency virus or occasionally in other immunocompromised hosts . There are scattered reports of H . cinaedi isolated from a variety of animal hosts, but to date only hamsters have been found to be a common natural reservoir . Microaerophillic cultures of feces from 5 of 16 asymptomatic rhesus monkeys (Macaca mulatta) (31%) were positive for a curved gram-negative rod . A polyphasic taxonomic approach was used to identify the organism as H . cinaedi . These results show that H . cinaedi frequently colonizes asymptomatic captive rhesus monkeys, which may serve as another potential reservoir for human infection.

Vaccine, 2002 Jun 21, 20(21-22), 2656 - 64
BHV-1 DNA vaccination: effect of the adjuvant RN-205 on the modulation of the immune response in mice; Zamorano P et al.; It is well documented that adjuvants improve the immune response generated by traditional viral vaccines, but less is known about the effects of adjuvants on the immune response elicited by DNA vaccines . In this study, we have investigated the use of RN-205 (immunomodulator containing a membrane rich in lipopolysaccharide from gram-negative bacteria) as an adjuvant and analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) . The comparison of the antibody response induced in mice by a mixture of the three different versions of DNA gD (membrane-anchored, secreted and cytosolic) formulated with or without RN-205 showed that the immunomodulator did not affect the total specific humoral response . The cellular immune response induced in mice immunized with vaccines plus RN-205 was higher than that obtained in mice vaccinated without RN-205, not only in the indexes of proliferation tests but in the number of IL-4 and gammaIFN secreting cells . When total spleen cells were marked with specific monoclonal antibodies against surface markers, a significant increase in the macrophage population of all the groups receiving RN-205 was observed . CD8 and CD4 positive cells were also increased but to a lesser extent . Our results indicate that the incorporation of RN-205 into DNA vaccines induces an increase of the cellular specific immune response in mice.

Clin Infect Dis, 2002 Jun 15, 34(12), 1600 - 6 Epub 2002 May 23.
Nosocomial bacteremia caused by antibiotic-resistant gram-negative bacteria in critically ill patients: clinical outcome and length of hospitalization; Blot S et al.; Population characteristics and outcomes were retrospectively compared for critically ill patients with nosocomial bacteremia caused by antibiotic-susceptible (AB-S; n=208) or antibiotic-resistant (AB-R; n=120) gram-negative bacteria . No significant differences in severity of illness and comorbidity factors were seen between groups . Patients with bacteremia caused by AB-R strains had a longer hospitalization before the onset of the bacteremia . The in-hospital mortality for patients with bacteremia caused by AB-S strains was 41.8%; for patients infected with AB-R strains, it was 45.0% (P=.576) . A multivariate survival analysis demonstrated that older age (P=.009), a high-risk source of bacteremia (abdominal and lower respiratory tract; P=.031), and a high acute physiology and chronic health evaluation II-related expected mortality (P=.032) were independently associated with in-hospital mortality (P<.05) . Antibiotic resistance in nosocomial bacteremia caused by gram-negative bacteria does not adversely affect the outcome for critically ill patients.

Pediatr Res, 2002 Jun, 51(6), 670 - 4
Extracellular release of bactericidal/permeability-increasing protein in newborn infants; Nupponen I et al.; Upon activation, polymorphonuclear leucocytes (PMN) release bactericidal/permeability-increasing protein, (BPI) from their azurophil granules . BPI selectively binds to the lipopolysaccharide (LPS) on gram-negative bacteria and induces their death . This study examined plasma BPI concentration levels in healthy newborns and in newborns with clinical sepsis, and the ability of PMN from preterm and term infants to release BPI . We also studied the release of myeloperoxidase (MPO), and the surface expression of adhesion molecule CD11b on PMN . In infants with clinical sepsis, plasma BPI concentration was higher, 27.8 microg/L {8.6-883; median (range)} (n = 11), than in healthy term infants 8.9 microg/L (3.9-179) (n = 17), and in adults 7.3 microg/L (0.7 -18.4) (n = 15); p = 0.014, Kruskal-Wallis . In preterm infants (n = 8), the ability of PMN to release BPI in vitro after stimulation with PMA was 8.8, in term infants it was 15.9 (n = 29; p > 0.05 vs . preterm infants) and in adults 23.4 ng/10(6) PMN (n = 15; p = 0.024 and p > 0.05 vs . preterm and term infants, respectively) . The corresponding values of MPO were 20.0 ng/10(6) (11.3-46.7) in preterms, 19.0 ng/10(6) (2.2-223.7) in terms, and 27.8 ng/10(6) (9.1-80.7) in adults; p = 0.67 between groups . In infants with clinical sepsis, CD11b level was higher, 292 RFU (234-403) than the basal CD11b expression levels in healthy newborn infants, 116 RFU (76-145); P = 0.0001 . FMLP-stimulated PMN CD11b expressions in preterm cord blood, 1071 RFU (552-1286) and in term cord blood, 918 (567-1472) were on the same level, but lower than that in adult blood, 1592 (973-1946); p < 0.001, ANOVA . Our findings suggest that in preterm infants the ability to release BPI is lower than in adults and term infants . These findings suggest that premature neonates have an impaired ability to mobilize BPI, possibly contributing to their marked susceptibility to infections with Gram-negative bacteria.

J Microbiol Methods, 2002 Aug, 50(3), 283 - 9
Limitations in the use of 3-hydroxy fatty acid analysis to determine endotoxin in mammalian samples; Szponar B et al.; 3-Hydroxy fatty acids (3-OH FAs) of 10-18-carbon chain lengths are constituents of the lipopolysaccharide of Gram-negative bacteria . These acids are used as chemical markers for determining endotoxin in environmental samples . The present communication addresses the question whether this type of analysis also would be applicable to mammalian samples . Low levels (6.1+/-1.6-94.0+/-23.2 pmol/ml) of the studied 3-OH FAs were detected in blood from both conventional and germ-free rats . The levels were considerably higher (0.0-1.06+/-0.17 nmol/mg) in livers . The amounts of the 3-OH FAs did not differ between the two groups of rats . All analyses were made by gas chromatography-tandem mass spectrometry (GC-MSMS) for unequivocal identification . The results illustrate a limitation in using 3-OH FA analysis to determine endotoxin in mammalian samples since these acids may represent not only endotoxin but also products from mammalian mitochondrial fatty acid beta-oxidation.

Oral Microbiol Immunol, 2002 Jun, 17(3), 143 - 9
Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans; Murakami Y et al.; Actinobacillus actinomycetemcomitans is a gram-negative bacterium strongly implicated in the pathogenesis of juvenile periodontitis . This periodontal pathogen synthesizes a leukotoxin that destroys human polymorphonuclear leukocytes (PMNs), and this toxin is thought to be responsible for the virulence of A . actinomycetemcomitans . It was therefore of interest to assess whether major virulence factors of periodontal pathogens were neutralized by salivary components . This study focuses on the effect of histatins, components of the nonimmune oral defense system, on leukotoxin activity . Leukotoxin was extracted with polymyxin B from freshly grown anaerobic cultures of A . actinomycetemcomitans strain Y4 . PMNs isolated from blood of healthy human volunteers were incubated in a cytotoxicity assay containing PMNs (10(7) cells/ml) and leukotoxin preparation (0-500 microg/ml) in Hanks' balanced salt solution at 37 degrees C for 0-120 min with or without synthetic histatin 5 (0-500 microM) . Cytotoxicity was measured by release of lactate dehydrogenase (LDH) at different time intervals . Histatin 5 neutralized the toxic effect of the leukotoxin preparation in a concentration-dependent manner, with an IC(50) value of 150 microM . When PMNs were preincubated with histatin 5 (300 microM), washed and subsequently exposed to leukotoxin, no protective effect was observed . This observation suggests a mechanism of inhibition whereby histatin 5 either directly neutralizes the leukotoxin or interferes with the leukotoxin-PMN interaction . The inhibitory effect of histatin 5 on leukotoxic activity may suggest a new biological function of histatins in the oral cavity as a naturally occurring secondary antibiotic.

Can J Microbiol, 2002 Apr, 48(4), 374 - 8
Evidence of quorum sensing in the rumen ecosystem: detection of N-acyl homoserine lactone autoinducers in ruminal contents; Erickson DL et al.; Acyl-homoserine lactone (AHL) based quorum-sensing systems are widespread among gram-negative bacteria, particularly in association with plants and animals . As yet, there have been no reports of AHL signaling in the anaerobic rumen environment, an ecosystem of great complexity in which cell-cell signaling is likely to occur . We detected multiple AHL autoinducers in the rumen contents of 6 out of 8 cattle fed a representative selection of diets . The signals were not associated with feed . Surprisingly, no pure cultures produced AHLs in vitro when grown under the laboratory conditions we tested . Our observations suggest that either (a) a factor specific to the rumen ecosystem is required for the rumen isolates we tested to produce AHLs or (b) a strain (or strains) that we were not able to culture but which grows to a high cell density in the rumen produces the AHLs we detected.

Arch Microbiol, 2002 Jun, 177(6), 468 - 74 Epub 2002 Apr 03.
Desulfobulbus mediterraneus sp . nov., a sulfate-reducing bacterium growing on mono- and disaccharides; Sass A et al.; A new sulfate-reducing bacterium, strain 86FS1, was isolated from a deep-sea sediment in the western Mediterranean Sea with sodium lactate as electron and carbon source . Cells were ovoid, gram-negative and motile . Strain 86FS1 contained b- and c-type cytochromes . The organism was able to utilize propionate, pyruvate, lactate, succinate, fumarate, malate, alanine, primary alcohols (C(2)-C(5)), and mono- and disaccharides (glucose, fructose, galactose, ribose, sucrose, cellobiose, lactose) as electron donors for the reduction of sulfate, sulfite or thiosulfate . The major products of carbon metabolism were acetate and CO(2), with exception of n-butanol and n-pentanol, which were oxidized only to the corresponding fatty acids . The growth yield with sulfate and glucose or lactate was 8.3 and 15 g dry mass, respectively, per mol sulfate . The temperature limits for growth were 10 degrees C and 30 degrees C with an optimum at 25 degrees C . Growth was observed at salinities ranging from 10 to 70 g NaCl l(-1) . Sulfide concentrations above 4 mmol l(-1) inhibited growth . The fatty acid pattern of strain 86FS1 resembled that of Desulfobulbus propionicus with n-14:0, n-16:1omega7, n-16:1 omega5, n-17:1 omega6 and n-18:1 omega7 as dominant fatty acids . On the basis of its phylogenetic position and its phenotypic properties, strain 86FS1 affiliates with the genus Desulfobulbus and is described as a new species, Desulfobulbus mediterraneus sp . nov.

Int J Artif Organs, 2002 Apr, 25(4), 249 - 60
Peritonitis in peritoneal dialysis; Voinescu CG et al.; Peritonitis, an infectious complication of peritoneal dialysis, continues to account for much of the morbidity associated with this techniques . The clinical presentation and laboratory data used in diagnosis the peritonitis, as well as its differential diagnosis will be reviewed in this article . The distribution of pathogens is an important outcome determinant, Gram-negative infections being associated with greater rates of catheter loss and higher death rates . Among the five routes of peritoneal contamination, intraluminal and periluminal contamination account for most of the infections . Due to the two prevention methods implemented in the care of the PD population, the incidence of peritonitis has decreased over the last two decades . The recommendations for empiric treatment of peritonitis have changed over the years, as more was learnt about antibiotic resistance and drug toxicity . Future research to address enteric peritonitis, as well as biocompatible dialysis solution or biocompatible catheter materials is needed to further reduce the incidence of PD peritonitis.

Infect Control Hosp Epidemiol, 2002 May, 23(5), 285 - 9
Application of environmental sampling to flexible endoscope reprocessing: the importance of monitoring the rinse water; Muscarella LF; The routine sampling of environmental surfaces within a healthcare facility is generally not recommended by the Centers for Disease Control and Prevention (CDC), the Association for the Advancement of Medical Instrumentation (AAMI), and several other healthcare organizations . There are a few circumstances, however, for which some organizations do recommend this practice . For instance, the CDC and the Association for Professionals in Infection Control and Epidemiology (APIC) recommend environmental sampling as clinically required during an outbreak investigation . The CDC and AAMI also recommend routine sampling of the rinse water used during hemodialyzer (but not endoscope) reprocessing . The rationale for this recommendation is based in part on reports of pyrogenic responses, patient infections, and bacteremia due to waterborne, gram-negative bacteria during hemodialysis . To determine whether the basis for this rationale might similarly apply to the rinse water used during endoscope reprocessing, the Food and Drug Administration's medical device reporting database, the endoscope reprocessing literature, and other sources were reviewed . The results of this review indicate that nosocomial outbreaks linked to endoscopes contaminated with gram-negative bacteria have been frequently reported . As a result, for several reasons, including to minimize the risk of patient infection due to gram-negative bacteria following endoscopy, this article recommends routine microbiologic sampling of the rinse water used during endoscope reprocessing.

Infect Control Hosp Epidemiol, 2002 May, 23(5), 239 - 43
Bloodstream infections in pediatric oncology outpatients: a new healthcare systems challenge; Smith TL et al.; OBJECTIVE: To investigate a perceived increase in central venous catheter (CVC)-associated bloodstream infections (BSIs) among pediatric hematology-oncology outpatients . DESIGN: A case-control study . SETTING: A pediatric hematology-oncology outpatient clinic at Fresno Children's Hospital . PATIENTS: Pediatric hematology-oncology clinic outpatients with CVCs at Fresno Children's Hospital between November 1994 and October 1997 . METHODS: A case-patient was defined as any hematology-oncology outpatient with a CVC-associated BSI at Fresno Children's Hospital from November 1996 to October 1997 (study period) without a localizable infection . To identify case-patients, we reviewed Fresno Children's Hospital records for all hematology-oncology clinic patients, those with CVCs and those with CVCs and BSIs . Control-patients were randomly selected hematology-oncology outpatients with a CVC but no BSI during the study period . Case-patient and control-patient demographics, diagnoses, caretakers, catheter types, catheter care, and water exposure were compared . RESULTS: Twenty-five case-patients had 42 CVC-associated BSIs during the study period . No significant increase in CVC-associated BSI rates occurred among pediatric hematology-oncology patients . However, there was a statistically significant increase in nonendogenous, gram-negative (eg, Pseudomonas species) BSIs during summer months (May-October) compared with the rest of the year . Case-patients and control-patients differed only in catheter type; case-patients were more likely than control-patients to have a transcutaneous CVC . Summertime recreational water exposures were similar and high in the two groups . CONCLUSIONS: Hematology-oncology clinic patients with transcutaneous CVCs are at greater risk for CVC-associated BSI, particularly during the summer . Caretakers should be instructed on proper care of CVCs, particularly protection of CVCs during bathing and recreational summer water activities, to reduce the risk of nonendogenous, gram-negative BSIs.

Mikrobiologiia, 2002 Mar-Apr, 71(2), 171 - 5
{Tolerance of soil bacterial complexes to salt shock}; Lapygina EV et al.; Investigations showed that bacteria present in soil are resistant to one-day exposure to a saturated solution of ammonium nitrate and can well develop when transferred to laboratory nutrient media . The evaluated number of bacteria in NH4NO3-treated soil samples was nearly the same as in native soil samples, while was 1.5-2.5 times smaller in the former than in the latter case when microbial succession in the soil samples was initiated by wetting them . Bacteria (particularly gram-negative ones) occurring at the early stages of succession were the most sensitive to salt stress . Bacteria in soil were found to be much more resistant to salt stress than the same bacteria isolated in pure cultures.

Microb Ecol, 2002 Mar, 43(2), 189 - 98 Epub 2002 Jan 02.
Lipid analysis of the response of a sedimentary microbial community to polycyclic aromatic hydrocarbons; Langworthy DE et al.; Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants that can, under proper conditions, be degraded by microorganisms . The responses of a riverine sedimentary microbial community to PAH contamination were examined using an integrated biochemical assay that yielded data on PAH concentration, total microbial biomass, and microbial community structure and were interpreted using perturbation theory and the subsidy-stress gradient . Microbial mineralization of naphthalene, anthracene, fluorene, and phenanthrene was observed 24 h after their addition to all sediments sampled and ranged from 0.9 to 16.3% in ambient sediments and from 14.8 to 35.8% in contaminated sediments . Total microbial biomass, determined by phospholipid phosphate, increased in response to intermediate PAH concentration and decreased at sites with the highest PAH concentration (p < 0.05) during seven out of nine (78%) seasonal sampling periods . The two sampling periods that were not statistically different followed periods of high water and cold temperatures . Phospholipid fatty acid analysis of microbial community structure analysis indicated that increases in the relative abundance of gram-negative aerobes and heterotrophic eukaryotes were responsible, in part, for these observed increases in total microbial biomass . These findings (increased degradation rates, increased biomass at intermediate PAH concentrations, and altered community structure) indicate that a component of the microbial community responded to PAH as a usable input and are consistent with the predictions of perturbation theory and a subsidy-stress gradient.

J Med Microbiol, 2002 Jun, 51(6), 491 - 4
Evaluation of liquid culture media to support growth of Mobiluncus species; Taylor-Robinson AW et al.; Mobiluncus curtisii and M . mulieris are anaerobic, gram-negative, motile curved rods isolated commonly from the vagina of women with bacterial vaginosis . Hitherto, there has been difficulty in isolating and growing these bacteria and little attention has been paid to growth in liquid media . Reasons for establishing the means of attaining optimal growth in such media include production of antigens for diagnostic and immunological studies and production of the soluble cytotoxin . In this study the efficacy of 12 liquid culture media in supporting growth was examined . M . mulieris (strain A198) multiplied > or =10-fold in only five media - Schaedler broth, Columbia blood broth (CBB), peptone-starch-dextrose (PSD) broth, brain-heart infusion plus arginine and spent tissue-culture medium . Similarly, M . curtisii (strain A98) multiplied > or =10-fold in only three media -Schaedler broth, CBB and PSD . Some strains of both bacterial species grew very poorly or not at all, in all the media tested . With an inoculum of > or =10(5)/ml, CBB, or PSD plus 10% horse serum, supported the growth of some strains of both bacterial species to 10(9) organisms/ml within 48 h, and viable bacteria persisted longer in some media (e.g., CBB) than in others . While variation in growth of Mobiluncus spp . may occur between one laboratory and another, these observations provide the basis for optimisation of a universal liquid culture medium that should facilitate production of antigens and cytotoxin.

Appl Microbiol Biotechnol, 2002 May, 58(6), 772 - 80 Epub 2002 Mar 08.
Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia; Neumann S et al.; The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides . Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence . Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene . The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa . The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3 . The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM . The natural function of Pam remains unclear . Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors . When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S . maltophilia . In the expression system, Pam made up about 31% of the total soluble cell protein . From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.

Neuroradiology, 2002 May, 44(5), 438 - 42 Epub 2002 Feb 08.
Dynamic changes during evacuation of a left temporal abscess in open MRI: technical case report; Bernays RL et al.; We demonstrate the usefulness of "near real-time" neuro-navigation by open MRI systems for guidance of stereotactic evacuation of intracranial abscesses . A 70-year-old patient was referred to our institution with an intracranial left temporal abscess . He presented with headache, senso-motor aphasia and mild right hemiparesis . The abscess (35 x 25 mm) was stereotactically evacuated under MRI guidance, and a recurrence of a daughter abscess was again evacuated on the 9th postoperative day . "Near real-time" imaging showed an indentation of the abscess wall of 11 mm along the trajectory . A thermosensitive MRI protocol demonstrated a higher temperature around the abscess capsule than in the brain tissue more distant to the capsule, demonstrating the inflammatory process . The patient had 6 weeks of antibiotic therapy for gram-negative bacteria and was discharged with improved clinical symptoms 5 weeks after admission . Follow-up CT 2 months postoperatively showed a complete resolution of the abscess . Open MRI-guided interventions with "near real-time" imaging demonstrate the anatomical changes during an ongoing procedure and can be accommodated for enhancing the overall precision of stereotactic procedures . Thermosensitive MRI protocols are capable of revealing temperature gradients around inflammatory processes.

Infect Immun, 2002 Jun, 70(6), 2995 - 3003
Lipopolysaccharide down regulates both scavenger receptor B1 and ATP binding cassette transporter A1 in RAW cells; Baranova I et al.; Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formation and has been suggested to be a proatherogenic factor . The mechanism of LPS induced cholesterol accumulation, however, is unclear . In this report, using the macrophage-like RAW 264.7 cell line, we provide experimental evidence that LPS's proatherogenic effects may at least in part reflect altered cholesterol metabolism . Data presented demonstrate that in a dose-dependent manner, LPS is able to down regulate the mRNA expression of the two primary high-density lipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80% . We also found that LPS treatment resulted in a significant decrease (to 20% of the control level) of the specific (125)I-HDL binding as well as in 50% inhibition of the HDL-mediated cholesterol efflux compared to untreated cells . In addition, we compared the potencies of various modified LPS preparations and demonstrated that the phosphorylated lipid A portion of LPS, which is highly conserved among gram-negative microorganisms, including Chlamydia, is primarily responsible for the effects of LPS on SR-B1 and ABCA1 expression . Inhibitors of NF-kappaB activation were observed to efficiently block the suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-kappaB . These data indicate that the LPS effects on cholesterol metabolism may contribute to the proatherogenic properties of LPS.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 189 - 95
Cytochromes c of Acidithiobacillus ferrooxidans; Yarzabal A et al.; The chemolithoautotrophic Gram-negative bacterium Acidithiobacillus ferrooxidans is versatile and can grow on a number of electron donors and acceptors . In the A . ferrooxidans ATCC 23270 genome, computer analysis identified 11 genes encoding putative cytochromes c . At least eight putative cytochromes c were differentiated on gels in ATCC 33020 cells grown on ferrous iron or sulfur . All these cytochromes were associated with the inner or the outer membranes . Lower levels of total cytochromes c were observed in sulfur- than in ferrous iron-grown cells . One cytochrome c was specific for sulfur conditions while three were specific for iron conditions, suggesting that cytochrome c synthesis is modulated depending on the electron donor.

J Biol Chem, 2002 Jul 19, 277(29), 26057 - 65 Epub 2002 May 02.
Cloning and characterization of a leucyl aminopeptidase from three pathogenic Leishmania species; Morty RE et al.; Aminopeptidases are emerging as exciting novel drug targets and vaccine candidates in parasitic infections . In this study, we describe for the first time an aminopeptidase from three highly pathogenic Leishmania species . Intronless genes encoding a leucyl aminopeptidase (lap) were cloned from Leishmania amazonensis, Leishmania donovani, and Leishmania major, which encoded 60-kDa proteins that displayed homology to leucyl aminopeptidases from Gram-negative bacteria, plants, and mammals . The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L . amazonensis . Lap assembled into catalytically competent 360-kDa hexamers and demonstrated potent amidolytic activity against synthetic aminopeptidase substrates containing leucine, methionine, and cysteine residues, representing the most restricted substrate specificity of any leucyl aminopeptidase described to date . Optimal activity was observed against L-leucyl-7-amido-4-methylcoumarin (k(cat)/K(m) approximately 63 s(-1) x mm(-1)) with a pH optimum of 8.5 . Leishmania Lap activity was inhibited by metal ion chelators and enhanced by divalent manganese, cobalt, and nickel cations, although only zinc was detected in the purified Lap by inductively coupled plasma atomic emission spectroscopy, indicating that zinc is the natural Lap cofactor . Activity was potently inhibited by bestatin and apstatin in a slow binding competitive fashion, with K(i)* values of 3 and 44 nm, respectively . Actinonin was a tight binding competitive inhibitor (K(i) approximately 1 nm), whereas arphamenine A (K(i) approximately 70 microm) and L-leucinol (K(i) approximately 100 microm) were non-tight binding competitive inhibitors . Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.

Clin Oral Implants Res, 2002 Feb, 13(1), 1 - 19
Infectious risks for oral implants: a review of the literature; Quirynen M et al.; The use of oral implants in the rehabilitation of partially and fully edentulous patients is widely accepted even though failures do occur . The chance for implants to integrate can for example be jeopardised by the intra-oral presence of bacteria and concomitant inflammatory reactions . The longevity of osseointegrated implants can be compromised by occlusal overload and/or plaque-induced peri-implantitis, depending on the implant geometry and surface characteristics . Animal studies, cross-sectional and longitudinal observations in man, as well as association studies indicate that peri-implantitis is characterised by a microbiota comparable to that of periodontitis (high proportion of anaerobic Gram-negative rods, motile organisms and spirochetes), but this does not necessarily prove a causal relationship . However, in order to prevent such a bacterial shift, the following measures can be considered: periodontal health in the remaining dentition (to prevent bacterial translocation), the avoidance of deepened peri-implant pockets, and the use of a relatively smooth abutment and implant surface . Finally, periodontitis enhancing factors such as smoking and poor oral hygiene also increase the risk for peri-implantitis . Whether the susceptibility for periodontitis is related to that for peri-implantitis may vary according to the implant type and especially its surface topography.

South Med J, 2002 May, 95(5), 542 - 4
Rhabdomyolysis of infectious and noninfectious causes; Blanco JR et al.; BACKGROUND: This study was done to determine variables associated with infectious rhabdomyolysis (IRM) . METHODS: In this retrospective case-control study, rhabdomyolysis (RM) was defined as a fivefold or greater elevation in creatine kinase (CK) levels with a muscle/brain (MB) fraction <5% . Patients with myocardial infarction or cerebrovascular accident or a recent history of surgery, trauma, or immobilization were excluded . RESULTS: We analyzed 52 cases of RM seen at our institution between 1992 and 2000; IRM was the most frequent cause (31%), most commonly respiratory tract infections (38%) . When a microorganism could be identified (50%), it was more often gram-negative (63%) . Patients with IRM were elderly and had fever and lower CK levels . Infectious rhabdomyolysis was associated with a higher morbidity but not with a higher risk of death . CONCLUSIONS: Infectious rhabdomyolysis is the main cause of RM and must be suspected in elderly patients with fever and low levels of CK.

Crit Care Med, 2002 May, 30(5 Suppl), S207 - 13
Innate immune recognition of lipopolysaccharide by endothelial cells; Henneke P et al.; OBJECTIVE: The endothelium is an active component of the innate immune response to bacterial invasion . Endothelial cells comprise mechanisms to recognize structural patterns expressed by pathogens and subsequently initiate the transcription of inflammatory genes . The purpose of this article is to summarize the molecular processes that underlie the endothelial innate immune response to microbial components, with a particular focus on responses to Gram-negative bacterial lipopolysaccharide . DATA SOURCES: Personal observations and review of the literature as revealed by the National Library of Medicine . DATA SUMMARY AND CONCLUSION: Endothelial cells recognize the presence of microbial components such as lipopolysaccharide via a receptor complex that contains at least three important cell surface components: CD14, Toll-like receptor-4, and MD-2 . CD14 and MD-2 exist as soluble receptor components and are thought to bind to both lipopolysaccharide and Toll-like receptor-4, whereas Toll-like receptor-4 itself is the transmembrane signal transducer . Single-point mutations in MD-2 or the cytoplasmic portion of Toll-like receptor-4 abrogate the response to lipopolysaccharide . The composition of this receptor and the recruitment and activation of various cytoplasmic proteins afford at least five levels of ligand specificity and suggest that there are at least as many potential therapeutic targets for Toll-like receptor-mediated inflammatory states, including sepsis.

J Bacteriol, 2002 Jun, 184(11), 3096 - 105
The ner gene of Photorhabdus: effects on primary-form-specific phenotypes and outer membrane protein composition; O'Neill KH et al.; The nematode-bacterium complex of Heterorhabditis-Photorhabdus is pathogenic to insect larvae . The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes . The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries . Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as ner, a regulatory gene in gram-negative bacteria and their phages . Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form . These and other pieces of evidence indicate that the endogenous ner gene is not responsible for the secondary phenotype, but that ner, when overexpressed, can repress expression of primary phenotypes at stationary phase . Inactivation of the endogenous ner gene in the primary form affected the outer membrane protein profile . A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the ner primary strain.

J Bacteriol, 2002 Jun, 184(11), 2969 - 77
Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10; Sluis MK et al.; Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO(2) to form acetoacetate . In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified . The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (alpha(2)beta(2)gamma(2) multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH(3)COCH(3) + CO(2) + ATP-->CH(3)COCH(2)COO(-) + H(+) + AMP + 2P(i)), and kinetic properties (K(m) for acetone, 8 microM; k(cat) = 45 min(-1)) . Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources . The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X . autotrophicus and sequence analysis of the R . capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (beta subunit), acxB (alpha subunit), and acxC (gamma subunit) . Transposon mutagenesis of X . autotrophicus revealed a requirement of sigma(54) and a sigma(54)-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression . A potential sigma(54)-dependent promoter 122 bp upstream of X . autotrophicus acxABC was identified . An AcxR gene homolog was identified 127 bp upstream of acxA in R . capsulatus, but this activator lacked key features of sigma(54)-dependent activators, and the associated acxABC lacked an apparent sigma(54)-dependent promoter, suggesting that sigma(54) is not required for expression of acxABC in R . capsulatus . These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria.

Crit Rev Oral Biol Med, 2001, 12(5), 399 - 413
Role of Treponema denticola in periodontal diseases; Sela MN; Among periodontal anaerobic pathogens, the oral spirochetes, and especially Treponema denticola, have been associated with periodontal diseases such as early-onset periodontitis, necrotizing ulcerative gingivitis, and acute pericoronitis . Basic research as well as clinical evidence suggest that the prevalence of T denticola, together with other proteolytic gram-negative bacteria in high numbers in periodontal pockets, may play an important role in the progression of periodontal disease . The accumulation of these bacteria and their products in the pocket may render the surface lining periodontal cells highly susceptible to lysis and damage . T . denticola has been shown to adhere to fibroblasts and epithelial cells, as well as to extracellular matrix components present in periodontal tissues, and to produce several deleterious factors that may contribute to the virulence of the bacteria . These bacterial components include outer-sheath-associated peptidases, chymotrypsin-like and trypsin-like proteinases, hemolytic and hemagglutinating activities, adhesins that bind to matrix proteins and cells, and an outer-sheath protein with pore-forming properties . The effects of T . denticola whole cells and their products on a variety of host mucosal and immunological cells has been studied extensively (Fig . 1) . The clinical data regarding the presence of T . denticola in periodontal health and disease, together with the basic research results involving the role of T . denticola factors and products in relation to periodontal diseases, are reviewed and discussed in this article.

Pediatr Transplant, 2002 Apr, 6(2), 132 - 5
Biliary stricture in living-related donor liver transplantation: management with balloon dilation; Schwarzenberg SJ et al.; Biliary stricture is a recipient graft complication, occurring late in the post-operative period, which appears to occur with increased frequency in living-related donor liver transplantation (LRD LTx) . We reviewed the experience at the University of Minnesota in managing a biliary complication of LRD LTx . Since January 1997, 13 LRD transplants have been performed using the technique of transplantation of the left lateral segments with a small portion of segment IV . All patients had hepaticojejunostomies using a Roux-en-Y loop . Of the 11 surviving patients, eight had evidence of cholangitis (Gram-negative sepsis, two patients; ascending cholangitis, three patients; or unexplained fever with elevated liver enzymes, three patients) 4-8 months after otherwise successful transplantation . Six of the patients underwent percutaneous transhepatic cholangiography (PTC) with demonstration of a stenosis at the site of the biliary anastomosis . Repeated dilation of the anastomosis led to resolution of the stenoses, normalization of liver enzymes, and prevention of further episodes of infection . No patient required revision of the hepaticojejunostomy . Computed axial tomography evidence of ductal stenosis may be subtle in this group of patients, but PTC is diagnostic . We suggest a high index of suspicion of biliary stricture in the LRD LTx population . Biliary dilation reduces the risk of life-threatening sepsis.

Arch Intern Med, 2002 May 13, 162(9), 1028 - 32
Relevance of mutations in the TLR4 receptor in patients with gram-negative septic shock; Lorenz E et al.; BACKGROUND: Septic shock remains a significant health concern worldwide, and despite progress in understanding the physiological and molecular basis of septic shock, the high mortality rate of patients with septic shock remains unchanged . We recently identified a common polymorphism in toll-like receptor 4 (TLR4) that is associated with hyporesponsiveness to inhaled endotoxin or lipopolysaccharide in humans . METHODS: Since TLR4 is a major receptor for lipopolysaccharide in mammals and gram-negative bacteria are the prevalent pathogen associated with septic shock, we investigated whether these specific TLR4 alleles are associated with a predisposition to a more severe disease outcome for patients with septic shock . We genotyped 91 patients with septic shock as well as 73 healthy blood donor controls for the presence of the TLR4 Asp299Gly and TLR4 Thr399Ile mutations . RESULTS: We found the TLR4 Asp299Gly allele exclusively in patients with septic shock (P =.05) . Furthermore, patients with septic shock with the TLR4 Asp299Gly/Thr399Ile alleles had a higher prevalence of gram-negative infections . CONCLUSION: Mutations in the TLR4 receptor may predispose people to develop septic shock with gram-negative microorganisms.

Nat Struct Biol, 2002 Jun, 9(6), 447 - 52
E . coli aconitase B structure reveals a HEAT-like domain with implications for protein-protein recognition; Williams CH et al.; The major bifunctional aconitase of Escherichia coli (AcnB) serves as either an enzymic catalyst or a mRNA-binding post-transcriptional regulator, depending on the status of its iron sulfur cluster . AcnB represents a large, distinct group of Gram-negative bacterial aconitases that have an altered domain organization relative to mitochondrial aconitase and other aconitases . Here the 2.4 A structure of E . coli AcnB reveals a high degree of conservation at the active site despite its domain reorganization . It also reveals that the additional domain, characteristic of the AcnB subfamily, is a HEAT-like domain, implying a role in protein protein recognition . This domain packs against the remainder of the protein to form a tunnel leading to the aconitase active site, potentially for substrate channeling.

Am J Respir Crit Care Med, 2002 May 1, 165(9), 1329 - 35
Diesel exhaust particles enhance lung injury related to bacterial endotoxin through expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1; Takano H et al.; Epidemiologic studies demonstrate acute and serious adverse effects of particulate air pollution on respiratory health, especially in people who are susceptible to bacterial infection . However, the underlying mechanism remains to be elucidated . To provide experimental evidence for the epidemiologic data, we determined the effects of diesel exhaust particles (DEP), major participants in particulate pollutants, on lung injury related to bacterial endotoxin in mice . Intratracheal instillation of DEPs synergistically enhanced lung injury related to endotoxin from gram-negative bacteria, which was characterized by neutrophil sequestration, interstitial edema, and alveolar hemorrhage . In the presence of endotoxin, DEPs further activated the nuclear translocation of p65 subunit of nuclear factor-kappaB (NF-kappaB) in the lung and increased the lung expression of intercellular adhesion molecule-1, interleukin-1beta, macrophage chemoattractant protein-1, keratinocyte chemoattractant (KC), macrophage inflammatory protein-1alpha, and Toll-like receptors . DEPs given alone increased the lung expression of Toll-like receptor 4 and the nuclear localization of p50 subunit of NF-kappaB . The combined exposure to DEPs and endotoxin decreased nuclear localization of CCAAT/enhancer binding protein beta . These results provide the first experimental evidence that DEPs enhance neutrophilic lung inflammation related to bacterial endotoxin . The enhancement is mediated by the induction of proinflammatory molecules, likely through the expression of Toll-like receptors and the activation of p65-containing dimer(s) of NF-kappaB, such as p65/p50.

J Pept Sci, 2002 Apr, 8(4), 144 - 50
Identification of single amino acid residues essential for the binding of lipopolysaccharide (LPS) to LPS binding protein (LBP) residues 86-99 by using an Ala-scanning library; Reyes O et al.; Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors . This process is thought to be of great importance in systemic inflammatory reactions such as septic shock . A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP . We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99 . Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding . In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction . Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect . The mutant Lys95 was the most active in blocking LPS binding to LBP . These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.

Mol Biotechnol, 2002 May, 21(1), 1 - 7
Cloning of anti-lPS factor cDNA from Tachypleus tridentatus, expression in Bombyx mori larvae and its biological activity in vitro; Wang DN et al.; In this article we report the cloning and expression of a cDNA encoding Tachypleus anti-lipopolysaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxins . First, two degenerate primers were designed based on the sequence homology of anti-LPS factors purified from different species of horseshoe crab . The total RNA was extracted from amebocytes of Tachypleus tridentatus . The cDNA was then obtained by using the RT-PCR methods . Second, the cDNA of Tachypleus anti-LPS factor (TALF) was expressed in Bombyx mori larvae using baculovirus expression system, which showed a yield of up to 600 mg/L . Last, we determined the biological activity of the recombinant proteins by LPS neutralization assay and bacteriostatic assay in vitro.

Microbiology, 2002 May, 148(Pt 5), 1543 - 51
Regulation of bacterial motility in response to low pH in Escherichia coli: the role of H-NS protein; Soutourina OA et al.; The effect of detrimental conditions on bacterial motility in Escherichia coli was investigated . Expression profiling of mutant E . coli strains by DNA arrays and analysis of phenotypic traits demonstrated that motility and low-pH resistance are coordinately regulated . Analysis of transcriptional fusions suggests that bacterial motility in response to an acidic environment is mediated via the control by H-NS of flhDC expression . Moreover, the results suggested that the presence of an extended mRNA 5' end and DNA topology are required in this process . Finally, the presence of a similar regulatory region in several Gram-negative bacteria implies that this mechanism is largely conserved.

Biochim Biophys Acta, 2002 May 3, 1562(1-2), 6 - 31
Protein-translocating outer membrane porins of Gram-negative bacteria; Yen MR et al.; Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized . In this report, these five porin families are analyzed from structural and phylogenetic standpoints . They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families . All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses . The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes . The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny . The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred . The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins.

Anal Chem, 2002 Apr 15, 74(8), 1798 - 804
Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay; Schilling EA et al.; A critical requirement for achieving a micro total analytical system for the analysis of cells and their constituent proteins is to integrate the lysis and fractionation steps on-chip . Here, an experimental microfluidic system integrating the lysis of bacterial cells and the extraction of a large intracellular enzyme, beta-galactosidase, is demonstrated . The beta-galactosidase is detected and quantified using a fluorogenic enzyme assay and a numerical model . While the focus is on the lysis of typical gram-negative bacterial cells (E . coli), the techniques described here could, in principle, be applied to a variety of different cell types.

Pediatr Int, 2002 Feb, 44(1), 5 - 11
Increased production of serum IgA-class antibody to lipid A in Kawasaki disease; Takeshita S et al.; BACKGROUND: The etiology of Kawasaki disease (KD) remains unknown . To investigate whether a conventional bacterial antigen is involved in the pathogenesis of KD, we studied the serum response to lipopolysaccharide (LPS) . METHODS: We measured the serum levels of IgG-, IgM- and IgA-class antibodies (Ab) to lipid A, a toxic site of LPS, using enzyme-linked immunosorbent assay in 20 patients with KD, 11 patients with Gram-negative bacterial infection (GNBI), 27 healthy children and 12 healthy adults . RESULTS: The serum levels of anti-lipid A IgG, IgM and IgA tended to increase with advancing age in healthy children older than 6 months of age . The mean level of anti-lipid A IgM in the acute phase of GNBI and the mean levels of anti-lipid A IgM and IgA in the acute phase of KD were found to increase significantly, in comparison to the age-matched controls . Furthermore, the mean level of anti-lipid A IgA also showed a significant increase from the acute to the subacute phases of KD . Regarding the IgA-subclass response, higher titers of anti-lipid A specific Ab were seen in the IgA2 subclass than in the IgA1 subclass . CONCLUSION: These findings indicate that KD patients demonstrate an intense response to lipid A in the IgA, especially IgA2-subclass, thus suggesting that an unusual activation of the mucosal immune response to a ubiquitous antigen derived from Gram-negative bacteria may be involved in the pathogenesis of KD.

J Pineal Res, 2002 May, 32(4), 231 - 6
Melatonin protects against gentamicin-induced nephrotoxicity in rats; Sener G et al.; Acute renal failure is a major complication of gentamicin (GEN), which is widely used in the treatment of gram-negative infections . A large body of in vitro and in vivo evidence indicates that reactive oxygen metabolites (or free radicals) are important mediators of gentamicin nephrotoxicity . In this study we investigated the role of free radicals in gentamicin-induced nephrotoxicity and whether melatonin, a potent antioxidant could prevent it . For this purpose female Sprague-Dawley rats were given intraperitoneally either gentamicin sulphate (40 mg/kg), melatonin (10 mg/kg), gentamicin plus melatonin or vehicle (control) twice daily for 14 days . The rats were decapitated on the 15th day and kidneys were removed . Blood urea nitrogen (BUN) and creatinine levels were measured in the blood and malondialdehyde (MDA) and glutathione (GSH) levels, protein oxidation (PO) and myeloperoxidase (MPO) activity were determined in the renal tissue . Gentamicin was observed to cause a severe nephrotoxicity which was evidenced by an elevation of BUN and creatinine levels . The significant decrease in GSH and increases in MDA levels, PO and MPO activity indicated that GEN-induced tissue injury was mediated through oxidative reactions . On the other hand simultaneous melatonin administration protected kidney tissue against the oxidative damage and the nephrotoxic effect caused by GEN treatment.

Graefes Arch Clin Exp Ophthalmol, 2002 Apr, 240(4), 329 - 30 Epub 2002 Mar 05.
Endophthalmitis caused by Moraxella osloensis; Berrocal AM et al.; PURPOSE: To report the clinical presentation, antibiotic sensitivities, and treatment outcomes of endophthalmitis caused by Moraxella osloensis . METHODS: Case series: retrospective review of the medical records of all patients treated for endophthalmitis at Bascom Palmer Eye Institute between 1 January 1991 and 31 December 2000 . RESULTS: During the study interval, 757 eyes were treated for endophthalmitis . Moraxella osloensis was isolated from three eyes of two patients (3/757, or 0.39%) . In all three eyes, the endophthalmitis was delayed-onset and bleb-associated; Moraxella osloensis was isolated on chocolate agar and 5% sheep's blood agar using a RapNH commercial Kit (by Remel) through an automated system (Vitek) . Like most gram-negative organisms, Moraxella was sensitive to ceftazidime, ciprofloxacin, and the aminoglycosides . Although vision at presentation was poor, both patients regained baseline vision after treatment with pars plana vitrectomy and injection of intravitreal antibiotics . CONCLUSIONS: To our knowledge, this is the first report of endophthalmitis caused by Moraxella osloensis . Unlike most series of delayed-onset, bleb-associated endophthalmitis the visual prognosis following treatment for endophthalmitis caused by Moraxella osloensis appears to be generally favorable.

Am J Kidney Dis, 2002 May, 39(5), 937 - 47
Endotoxin removal by direct hemoperfusion with an adsorbent column using polymyxin B-immobilized fiber ameliorates systemic circulatory disturbance in patients with septic shock; Uriu K et al.; Direct hemoperfusion (DHP) with an adsorbent column using polymyxin B-immobilized fiber (PMX-F) has been shown to improve the state of shock in patients with septic shock . However, no evidence has been presented for a direct link between endotoxin removal by DHP with PMX-F and improvement in septic shock . We retrospectively analyzed clinical profiles of 24 patients with septic shock (16 patients, gram-negative; 8 patients, non-gram-negative septic shock) who underwent DHP with PMX-F . Patients with gram-negative septic shock were characterized by hyperdynamic circulation . DHP with PMX-F reduced blood endotoxin concentrations and ameliorated shock, with an improvement in hyperdynamic circulation in patients with gram-negative septic shock . Mean arterial pressure also was elevated after therapy in patients with non-gram-negative septic shock, but systemic hemodynamics were unaffected . Regardless of the causative microorganism, patients with endotoxemia (blood endotoxin level > 10 pg/mL) showed hyperdynamic shock, and DHP with PMX-F reduced blood endotoxin levels and ameliorated hyperdynamic circulation, whereas patients without endotoxemia showed features of shock without hyperdynamic circulation, and DHP with PMX-F ameliorated shock without affecting cardiac performance . In patients with gram-negative septic shock, blood endotoxin concentration correlated positively with cardiac output and negatively with systemic vascular resistance before DHP therapy . Reduction in blood endotoxin concentration by DHP therapy positively correlated with the reduction in cardiac output . Our findings indicate that the improvement in hyperdynamic circulation was related directly to endotoxin removal by the PMX-F column, and endotoxin has an important role in the development of hyperdynamic circulation in patients with gram-negative septic shock .

Pediatr Res, 2002 May, 51(5), 559 - 63
Role of OmpA and IbeB in Escherichia coli K1 invasion of brain microvascular endothelial cells in vitro and in vivo; Wang Y et al.; Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis, but it is incompletely understood how E . coli K1 crosses the blood-brain barrier . We have previously identified several E . coli determinants contributing to invasion of brain microvascular endothelial cells (BMEC) in vitro, which include OmpA and IbeB . In the present study, we constructed a mutant (E98) by deleting only OmpA (isogenic OmpA deletion mutant) from E . coli K1 strain RS218 (018:K1:H7) and also an isogenic OmpA deletion mutant from the ibeB-deleted mutant (IB7D5) of strain RS218 . As expected, the ompA and ibeB deletion mutants, E98 and IB7D5, respectively, were less invasive in BMEC in vitro compared with the parent strain . More importantly, their abilities to penetrate the blood-brain barrier were significantly less than those of the parent strain in the experimental hematogenous E . coli meningitis model . The combined ompA- and ibeB-deleted mutant, however, behaved similarly compared with its single-gene deletion mutants (E98 and IB7D5) in its ability to invade BMEC in vitro and to penetrate into the CNS in vivo . These findings indicate that OmpA and IbeB are the important determinants contributing to E . coli K1 crossing of the blood-brain barrier, but their contributions are not additive . Additional studies are needed to understand the reasons for no additive effect with OmpA and IbeB in E . coli K1 penetration into the CNS.

Clin Rev Allergy Immunol, 2002 Apr, 22(2), 189 - 204
Regulation of mast cell-mediated innate immunity during early response to bacterial infection; Malaviya R et al.; Although the area of research on the role of MCs in innate immunity is relatively new, a number of studies that are reviewed here provide substantial evidence that MCs play a critical role in host immune defense against gram-negative bacteria . The studies show that mast cells have the ability to recognize and engulf bacteria and they release a number of inflammatory mediators including interleukin (IL)-4, IL-6, IL-10, TNF alpha, and leukotrienes in response to bacterial challenge . MC-derived TNF alpha and leukotrienes are shown to be important for bacterial clearance and early recruitment of phagocytic help at the site of infection . Studies directed at elucidating the molecular mechanisms associated with mast cell recognition of bacteria and subsequent events leading to mast cell mediator release revealed that GPI anchored CD48 molecule present on the cell surface of mast cells serves as a receptor for the bacterial adhesion molecule, FimH . The ligation of CD48 receptor by FimH-expressing bacteria results in bacterial uptake into caveolar chambers . This distinct mechanism of bacterial uptake promotes bacterial survival inside the cytosol of the mast cells . Although the exact mechanism(s) of how MC-dependent inflammatory responses are regulated is currently not known, recent studies have shown that complement, CD11 beta/CD18 (Mac-1) and protein tyrosine kinase JAK3, and TLR4 are important for the full expression of MC-dependent innate immunity in mice.

Am J Respir Cell Mol Biol, 2002 May, 26(5), 572 - 8
Macrophages are necessary for maximal nuclear factor-kappa B activation in response to endotoxin; Koay MA et al.; To define the role of macrophages in regulating the lung's response to Escherichia coli endotoxin (lipopolysaccharide {LPS}), depletion of macrophages was accomplished by administration of dichloromethylene diphosphonate (clodronate) delivered via intratracheal (i.t.) and/or intravenous (i.v.) routes . Clodronate reduced the number of macrophages in lung lavage 48 h after either i.t . or i.v . administration, but combined i.t . + i.v . clodronate achieved the most profound depletion (90%) . Although i.t . clodronate alone had little effect on the evolution of lung inflammation, combined i.t . + i.v . clodronate treatment decreased neutrophilic alveolitis 4 h after exposure to aerosolized LPS by 80% compared with mice treated with empty liposomes . This decrease was associated with impaired activation of nuclear factor (NF)-kappa B and lower concentrations of tumor necrosis factor (TNF)-alpha in lung lavage fluid . Combined i.t . + i.v . clodronate markedly reduced lung NF-kappa B activation and the intensity of neutrophilic alveolitis after intraperitoneal (i.p.) LPS; however, i.v . clodronate alone had no effect on NF-kappa B activation in either liver or lung tissue or the development of neutrophilic alveolitis . We conclude that generalized macrophage depletion reduces NF-kappa B activation, generation of cytokines, and neutrophilic lung inflammation in response to gram negative bacterial endotoxin . These findings define the role of the macrophage as a critical component for initiation of the NF-kappa B-dependent innate immune response.

Mol Biol (Mosk), 2002 Mar-Apr, 36(2), 261 - 7
{Mobile gene cassettes and DNA integration elements }; Kovalevskaia NP; Integrons are unique natural systems for capturing and spreading the antibiotic resistance genes among Gram-negative bacteria . Gene transfer into small genomes and into plasmids is via site-specific recombination . Integrons act as receptors of antibiotic resistance cassettes . There are known more than 50 cassettes conferring resistance to beta-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptomycin, and other antibiotics . The structure of integrons and of gene cassettes are described and the mechanisms of capture, mobilization, and expression of cassettes considered.

J Virol, 2002 May, 76(10), 4866 - 72
The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry; Grahn AM et al.; The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside . The virus membrane and several structural proteins are involved in phage DNA entry . In this work we identified a new infectivity protein of PRD1 . Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction . The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection . In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form . Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.

Mol Microbiol, 2002 Apr, 44(1), 271 - 81
Quantification of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA; Higgs PI et al.; The TonB-dependent energy transduction system couples cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes across the outer membrane in Gram-negative bacteria . In Escherichia coli, the primary players known in this process to date are: FepA, the TonB-gated transporter for the siderophore enterochelin; TonB, the energy-transducing protein; and two cytoplasmic membrane proteins with less defined roles, ExbB and ExbD . In this study, we report the per cell numbers of TonB, ExbB, ExbD and FepA for cells grown under iron-replete and iron-limited conditions . Under iron-replete conditions, TonB and FepA were present at 335 +/- 78 and 504 +/- 165 copies per cell respectively . ExbB and ExbD, despite being encoded from the same operon, were not equimolar, being present at 2463 +/- 522 and 741 +/- 105 copies respectively . The ratio of these proteins was calculated at one TonB:two ExbD:seven ExbB under all four growth conditions tested . In contrast, the TonB:FepA ratio varied with iron status and according to the method used for iron limitation . Differences in the method of iron limitation also resulted in significant differences in cell size, skewing the per cell copy numbers for all proteins.

Aliment Pharmacol Ther, 2002 Apr, 16 Suppl 2, 217 - 28
Clarithromycin increases the release of heat shock protein B from Helicobacter pylori; Tsuzuki T et al.; BACKGROUND: Clarithromycin (CAM) may have certain indirect effects on Helicobacter pylori (H . pylori) other than its inhibitory activity on bacterial growth, as indicated in other infections with Gram-negative micro-organisms . In the present study, we examined the effects of lower concentrations of CAM on the release of heat shock protein B (HspB), one of the major antigenic proteins from H . pylori cells, as well as the changes in humoral immune response and histological degree of antral gastritis in patients who received eradication therapy with CAM . METHODS: The H . pylori strain 26695 and three CAM-resistant clinical isolates were cultured in broth with and without CAM (2-500 ng/mL) . Expression of H . pylori proteins was examined by two-dimensional (2D)-electrophoresis followed by N-terminal amino acid sequencing . Changes in host immune response and histological degree of antral gastritis were monitored in patients with peptic ulcer disease who received H . pylori eradication therapy . RESULTS: 2D electrophoresis showed 26 spots in extracellularly released proteins with different profiles from those in cytoplasmic proteins . The release of HspB increased after incubation with CAM (30-500 ng/mL) in all three H . pylori clinical isolates tested . Patients with failed H . pylori eradication after triple therapy with CAM, but not those with failed eradication after dual therapy without CAM, showed an increase in serum IgG1 and IgG2 antibodies against HspB along with a decrease in the degree of neutrophil and H . pylori colonization density in tissue sections . CONCLUSIONS: CAM may induce a humoral immune response against H . pylori and a decrease in gastric mucosal inflammation through up-regulation of the release of HspB from the bacteria in infected patients.

Blood, 2002 May 1, 99(9), 3411 - 8
Population depletion activates autonomous CD154-dependent survival in biopsylike Burkitt lymphoma cells; Challa A et al.; Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues . Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control . Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor . Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter . Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting . Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached . These data reveal an auto-inducible survival mechanism seemingly regulated through the monitoring of population size, a process somewhat akin to that of "quorum sensing" among gram-negative bacteria in which diffusible molecules provide a means of communication to coordinate gene expression with population density . This process could be activated as cells discern depletions in their community or when deprived of signals otherwise furnished within an appropriate environmental niche.

Life Sci Space Res, 1977, 15, 207 - 12
Medically important micro-organisms recovered from Apollo-Soyuz Test Project (ASTP) crew members; Taylor GR et al.; The possibility of significant alterations in the microbial populations inhabiting the integument and upper respiratory tract of space flight crew members has been proposed by various authors . The Apollo-Soyuz Test Project (ASTP), a unique space flight in which two teams of crew members from different geographical areas joined in space, presented an unusual opportunity to evaluate in-flight cross-contamination and other anomalous behavior of the microbial populations . Accordingly, the medically important microbes recovered from the five (3 USA and 2 USSR) ASTP crew members before and after flight were evaluated in relation to specific theoretical alterations . The phenomenon of "simplification", in which the number of different types of species is reduced during spaceflight, did not occur within the population of medically important micro-organisms . Spontaneous post-flight illness due to infectious agents ("Microbial Shock") was also absent . Intracrew transfer of pathogens was established, but intercrew transfer was absent . Dysbacteriosis, in which sampled areas are flooded with unusually large numbers of a single type of micro-organism, was not demonstrated although there was a significant increase in the incidence of gram-negative rods in the oral cavities of the two cosmonauts . The importance of all of these and other observations of the medically important micro-organisms recovered from ASTP crew members are explored with respect to past and future space flights.

Appl Microbiol Biotechnol, 2002 Apr, 58(5), 663 - 8 Epub 2002 Feb 08.
Isolation and characterization of a new succinic acid-producing bacterium, Mannheimia succiniciproducensMBEL55E, from bovine rumen; Lee PC et al.; A novel succinic acid-producing bacterium was isolated from bovine rumen . The bacterium is a non-motile, non-spore-forming, mesophilic and capnophilic gram-negative rod or coccobacillus . Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to the recently reclassified genus Mannheimia as a novel species, and has been named Mannheimia succiniciproducens MBEL55E . Under 100% CO(2) conditions, it grows well in the pH range of 6.0-7.5 and produces succinic acid, acetic acid and formic acid at a constant ratio of 2:1:1 . When M . succiniciproducensMBEL55E was cultured anaerobically in medium containing 20 g l(-1) glucose as carbon source, 13.5 g l(-1) of succinic acid was produced.

Eur Cytokine Netw, 2002 Jan-Mar, 13(1), 86 - 91
Mice lacking alpha(2)-macroglobulin show an increased host defense against Gram-negative bacterial sepsis, but are more susceptible to endotoxic shock; Hochepied T et al.; The onset of an acute phase response is one of the initial steps in the defense against an infectious organism . Alpha(2)-macroglobulin (alpha(2)M), an acute phase protein in most mammalian species, is known to have a broad antiprotease activity, but it can also bind a number of growth factors, cytokines, ions and lipid factors . We have shown that alpha(2)M-deficient (MAM-/-) mice are more resistant to a lethal Gram-negative infection compared to control mice . This increased resistance was reflected in significantly higher body temperatures, compared to control mice, during the infection as well as in a prolonged and increased survival . Moreover, the clearance of bacteria in MAM-/- mice was significantly more efficient than in control mice . On the other hand, MAM-/- mice were more susceptible to endotoxin . An LD(100) challenge with endotoxin in MAM-/- mice was not lethal for control mice . Our data suggest that alpha(2)M plays a dual role during an acute phase response . In the establishment of a lethal Gram-negative infection, leading to sepsis and septic shock, it has a mediating role by hampering the efficient clearance of bacteria . During endotoxic shock, however, alpha(2)M has a rather protective function.

Nat Struct Biol, 2002 May, 9(5), 343 - 7
Structural basis of translational control by Escherichia coli threonyl tRNA synthetase; Torres-Larios A et al.; Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon . The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding . The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface . The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.

J Biol Chem, 2002 Jun 21, 277(25), 22414 - 20 Epub 2002 Apr 12.
Gram-negative flagellin-induced self-tolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5; Mizel SB et al.; Flagellin from a number of Gram-negative bacteria induces cytokine and nitric oxide production by inflammatory cell types . In view of the evidence that flagellin responsiveness is subject to modulation, we explored the possibilities that a prior exposure to flagellin might result in a state of reduced flagellin responsiveness or tolerance and that lipopolysaccharide (LPS) and flagellin may induce a state of cross-tolerance to each other . Our results demonstrate that a prior exposure to flagellin results in a subsequent state of flagellin tolerance in human monocytes, THP1 cells, Jurkat cells, and COS-1 cells . Tolerance occurs within 2 h after addition of flagellin and does not require protein synthesis . Flagellin did not induce tolerance to LPS in monocytes and THP1 cells; however, LPS treatment of monocytes and THP1 cells resulted in a state of flagellin cross-tolerance . Flagellin-induced self-tolerance is not the result of a decrease in the steady-state level of toll-like receptor 5 (TLR5) or interleukin-1 receptor associated kinase (IRAK), but it is associated with a block in the release of IRAK from the TLR5 complex in flagellin-tolerant cells . Release is essential for IRAK activity because the TLR5-associated IRAK lacks kinase activity . LPS-induced cross-tolerance to flagellin is also associated with a block in IRAK release from TLR5 . These results provide evidence for a novel mechanism of TLR signaling control.

Infect Immun, 2002 May, 70(5), 2535 - 43
Recombinant Ochrobactrum anthropi expressing Brucella abortus Cu,Zn superoxide dismutase protects mice against B . abortus infection only after switching of immune responses to Th1 type; He Y et al.; The members of the genus Brucella are gram-negative, facultatively intracellular bacterial pathogens that cause brucellosis in many animal species and humans . Although live, attenuated vaccines are available to protect several animal species from the disease, there is no safe and effective vaccine for human use . Here we report that a bacterium that is closely related to Brucella species, Ochrobactrum anthropi, can be used as a vaccine vector for the delivery of Brucella antigens to mice, leading to the elicitation of protective immunity against brucellosis . Brucella abortus Cu,Zn superoxide dismutase (SOD), a protective Brucella antigen, was expressed in large amounts in O . anthropi strain 49237 by use of the broad-host-range plasmid pBBR1MCS . Neither O . anthropi strain 49237 nor the recombinant O . anthropi strain 49237SOD, expressing B . abortus Cu,Zn SOD, provided protection against virulent Brucella infection in mice . Analysis of immune responses indicated that strains 49237 and 49237SOD stimulated a mix of Th1 and Th2 type responses in the mice . After the immune response was switched to a Th1-biased response by addition of oligonucleotides containing unmethylated CpG motifs, both O . anthropi strain 49237 and the recombinant O . anthropi strain 49237SOD induced protection in mice . However, the protection conferred by strain 49237SOD was significantly better than that induced by the parental strain, 49237.

Infect Immun, 2002 May, 70(5), 2297 - 303
DsbA and DsbC are required for secretion of pertussis toxin by Bordetella pertussis; Stenson TH et al.; The Dsb family of enzymes catalyzes disulfide bond formation in the gram-negative periplasm, which is required for folding and assembly of many secreted proteins . Pertussis toxin is arguably the most complex toxin known: it is assembled from six subunits encoded by five genes (for subunits S1 to S5), with 11 intramolecular disulfide bonds . To examine the role of the Dsb enzymes in assembly and secretion of pertussis toxin, we identified and mutated the Bordetella pertussis dsbA, dsbB, and dsbC homologues . Mutations in dsbA or dsbB resulted in decreased levels of S1 (the A subunit) and S2 (a B-subunit protein), demonstrating that DsbA and DsbB are required for toxin assembly . Mutations in dsbC did not impair assembly of periplasmic toxin but resulted in decreased toxin secretion, suggesting a defect in the formation of the Ptl secretion complex.

EMBO J, 2002 Apr 15, 21(8), 1909 - 15
The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ; Li CM et al.; The type III secretion system (TTSS) is an essential requirement for the virulence of many Gram-negative bacteria infecting plants, animals and man . Pathogens use the TTSS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cell, where the effectors subvert host defences . Plant pathogens have to translocate their effector proteins through the plant cell wall barrier . The best candidates for directing effector protein traffic are bacterial appendages attached to the membrane-bound components of the TTSS . We have investigated the protein secretion route in relation to the TTSS appendage, termed the Hrp pilus, of the plant pathogen Pseudomonas syringae pv . tomato . By pulse expression of proteins combined with immunoelectron microscopy, we show that the Hrp pilus elongates by the addition of HrpA pilin subunits at the distal end, and that the effector protein HrpZ is secreted only from the pilus tip . Our results indicate that both HrpA and HrpZ travel through the Hrp pilus, which functions as a conduit for the long-distance translocation of effector proteins.

J Microbiol Immunol Infect, 2002 Mar, 35(1), 1 - 11
Antibiotics: action and resistance in gram-negative bacteria; Siu LK; Therapeutic control of beta-lactamase-producing bacteria has been a major clinical problem in the past 40 years . Gram-negative bacteria are most often resistant to antibiotics as a result of the acquisition of resistant genes or gene mutation . Studies have shown that newly developed antibiotics will shortly fail to be active against the bacteria because of the emergence of resistance . Some resistant bacteria have been found to exist even before the antibiotic was developed . Selective pressure by the antibiotic is, therefore, one of the major factors to explain the increase of resistance . Recently, numerous resistant mechanisms that differ in their substrate profiles have been described at increasing frequencies . The inappropriate use of new antibiotics with extended spectrum further complicated the problem . Because resistance is a largely unavoidable consequence of widespread use of antibiotics, it is crucial that the use of drugs is selective by exercising prudent judgment and not excessive . The actual prevalence of resistance should be continuously monitored each year . Caution should be paid to the direct extrapolation of study results from other geographic areas, because the local prevalence of resistance is unlikely to be identical to those reported elsewhere . The impact of resistance to an antibiotic and its specific mechanisms, including transmissibility, should also be carefully studied . Such information may help in designing strategies for maximizing the therapeutic usefulness of drugs and minimizing the emergence of resistance.

World J Surg, 2002 Jul, 26(7), 790 - 8 Epub 2002 Apr 15.
Molecular biology of endotoxin antagonism; Lazaron V et al.; The development of new therapies for the treatment of gram-negative bacterial sepsis has been the focus of extensive investigation . Molecular and cellular biologic techniques have led to important advances, including (1) identification of naturally occurring lipopolysaccharide (LPS)-binding proteins; (2) generation of novel LPS-binding antibodies, proteins, and peptides; and (3) characterization of the molecular determinants of LPS binding . In composite, these advances have facilitated comprehension of the manner in which gram-negative bacterial infection and concurrent endotoxemia exert detrimental effects on the mammalian host . The purpose of this review was to examine recent information regarding molecular determinants of LPS binding and the initial cellular signal transduction events, which lead to the local and systemic cytokine response and sepsis syndrome . Concurrently, the status of studies examining the effects of various endotoxin antagonists is reviewed . Data from these studies provide an indication of potential sites for therapeutic intervention as well as increasingly detailed understanding of the molecular mechanism of endotoxin antagonism . Taken together, these advances can be expected to further the development of the next generation of novel, adjuvant therapies for the treatment of sepsis syndrome caused by gram-negative bacterial infection and endotoxemia.

Vet Immunol Immunopathol, 2002 May, 86(1-2), 115 - 24
Recombinant bovine soluble CD14 sensitizes the mammary gland to lipopolysaccharide; Wang Y et al.; Standard therapies including administration of potent antibiotics, aggressive fluid resuscitation and metabolic support have not been successful in relieving symptoms and reducing mortality associated with acute coliform mastitis . It is important to understand the pathophysiological response of the mammary gland to coliform infections when designing preventive or therapeutic regimens for controlling coliform mastitis . Our laboratory has previously shown that macrophages and polymorphonuclear neutrophils in milk express CD14 on their cell surface . In this study, we found that soluble CD14 (sCD14) is present in milk whey as a 46kDa protein reacted with anti-ovine CD14 antibody . Additional functional studies found that: (1) under serum-free condition, complexes of LPS-recombinant bovine soluble CD14 (rbosCD14) induced activation of mammary ductal epithelial cells (as measured by changes in interleukin-8 (IL-8) mRNA level by competitive RT-PCR) at low concentrations of LPS after 6 or 24h incubation (1-1000ng/ml), whereas LPS alone did not induce activation of mammary ductal epithelial cells at the same concentrations, and (2) intramammary injection of low concentrations of LPS did not increase concentration of leukocytes in milk . In contrast, LPS-rbosCD14 complex containing the same concentration of LPS increased the concentration of leukocytes in the injected mammary gland at 12 and 24h post-injection . These results indicate that rbosCD14 sensitizes mammary epithelial cells to low concentrations of LPS in vitro and in vivo . Endogenous sCD14 in milk may be important in initiating host responses to Gram-negative bacterial infections.

J Arthroplasty, 2002 Apr, 17(3), 384 - 7
Brucellosis as a cause of septic loosening of total hip arthroplasty; Ortega-Andreu M et al.; Infection after a total hip arthroplasty is a severe complication and is associated with a high incidence of morbidity . We describe a case of late infection, 5 years after the implantation of a cemented hip prosthesis . The infection was caused by gram-negative Brucella melitensis and occurred in a 63-year-old man who owned cattle . As far as we know, such a complication has never been published before.

J Immunol, 2002 Apr 15, 168(8), 3974 - 82
Role of NADPH oxidase in the mechanism of lung neutrophil sequestration and microvessel injury induced by Gram-negative sepsis: studies in p47phox-/- and gp91phox-/- mice; Gao XP et al.; We addressed the role of O(2) generated by the NADPH oxidase complex in the mechanism of polymorphonuclear leukocyte (PMN) accumulation and transalveolar migration and lung microvascular injury . Studies were made in mice lacking the p47(phox) and gp91(phox) subunits of NADPH oxidase (p47(phox-/-) and gp91(phox-/-)) in which PMN are incapable of the respiratory burst . The mice were challenged i.p . with live Escherichia coli to induce sepsis . We observed time-dependent increases in PMN sequestration and migration from 1 to 6 h after challenge with 2 x 10(8) E . coli . The responses in knockout mice were greater post-E . coli challenge compared with control mice; i.e., transalveolar PMN migration post-E . coli challenge increased by approximately 50% in the null mice above values in wild type . The increased PMN infiltration was associated with decreased lung bacterial clearance . The generation of the chemoattractant macrophage-inflammatory protein-2 in lung tissue was greater in NADPH oxidase-defective mice after E . coli challenge than control mice; moreover, macrophage-inflammatory protein-2 Ab pretreatment prevented the PMN infiltration . We also observed that E . coli failed to increase lung microvascular permeability in p47(phox-/-) and gp91(phox-/-) mice despite the greater lung PMN sequestration . Thus, O(2) production is required for the induction of sepsis-induced lung microvascular injury . We conclude that NADPH oxidase-derived O(2) generation has an important bactericidal role, such that an impairment in bacterial clearance in NADPH oxidase-defective mice results in increased chemokine generation and lung tissue PMN infiltration.

Am J Respir Crit Care Med, 2002 Apr 1, 165(7), 911 - 5
Endotoxin activity and inflammatory markers in the airways of young patients with cystic fibrosis; Muhlebach MS et al.; Chronic endobronchial infection frequently caused by gram-negative organisms and an increased, neutrophil-dominated inflammation are characteristics of cystic fibrosis (CF) . The present study examines endotoxin levels in bronchoalveolar lavage fluids of CF versus non-CF (N) control children, and correlates these with the inflammatory markers interleukin-8 and neutrophils . Fifty-five patients with CF and 56 patients without CF between the ages of 0.04 to 13.25 years were included . Infection, defined as a bacterial count above 50,000 cfu/ml, was present in 27 CF and 25 N patients . Endotoxin levels were not different between patients with and without CF (infected: 74.9 +/- 12.1 EU/ml versus 51.4 +/- 12.5 EU/ml, p = 0.16; noninfected: 5.9 +/- 4.8 EU/ml versus 11.1 +/- 4.3 EU/ml, p = 0.28) . Endotoxin activity correlated to the number of gram-negative organisms in CF and N patients, and endotoxin activity per bacterial colony forming unit did not differ with various gram-negative species . Both interleukin-8 and neutrophils were positively correlated with endotoxin, but this slope was shifted toward higher levels of inflammation in CF patients . We conclude that it is unlikely that higher levels of endotoxin in the absence of viable bacteria explain the increased inflammatory response in CF.

Curr Opin Microbiol, 2002 Apr, 5(2), 173 - 8
Transcriptional, chemosensory and cell-contact-dependent regulation of type IV pilus expression; Winther-Larsen HC et al.; Expression of Type IV pili (Tfp), multifunctional surface appendages expressed by Gram-negative species of medical and environmental significance, has previously been shown to be regulated by consensus two-component systems . Elucidation of their unique biogenesis pathway and the dynamics of pilus growth and retraction involved in motility have revealed a novel step at which regulation might be imposed . Studies of Tfp expression following adherence to host tissue clearly demonstrate regulation by modulation of the retraction process . In addition, a large set of components related to flagellar chemosensory pathways has been shown to influence Tfp expression levels in many species . Like their flagellar counterparts, the Tfp-dedicated homologues are proposed to function by regulating motor function . Rather than dictating the switch frequencies of organelle rotation, however, they are hypothesized to control the rates of fiber extrusion and retraction.

Curr Opin Microbiol, 2002 Apr, 5(2), 166 - 72
Regulation of type III secretion systems; Francis MS et al.; Type III secretion systems are utilised by numerous Gram-negative bacteria to efficiently interact with a host . Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family . The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues . Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers . Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 445 - 56
Classification of three airborne bacteria and proposal of Hymenobacter aerophilus sp . nov; Buczolits S et al.; Three aerobic, gram-negative, rod-shaped, non-spore-forming, red-pigmented, airborne bacteria (I/26-Cor1T, I/32A-Cor1 and I/74-Cor2) collected in the Museo Correr (Venice, Italy) were investigated to determine their taxonomic status by analysing their biochemical, physiological and chemotaxonomic features and the G+C content of genomic DNA and by comparing their genomic fingerprints . Additionally, the almost complete 16S rRNA gene sequence of strain I/26-Cor1T was analysed . The three strains were nearly identical in their morphological, physiological, biochemical and chemotaxonomic properties . The strains contained a menaquinone system with the predominant menaquinone MK-7 and a fatty acid profile with C15:0 anteiso, C15:0 iso and C16:1 predominant . Phosphatidylethanolamine and several unidentified lipids were detected in the polar lipid profiles . The polyamine pattern consisted of sym-homospermidine as the major compound . meso-Diaminopimelic acid was found as the characteristic cell-wall diamino acid . The DNA base composition of the three strains ranged from 60 to 63 mol% G+C . Phylogenetically, strain I/26-Cor1T was most closely related to Hymenobacter actinosclerus (95.8% 16S rRNA gene sequence similarity) . Physiological and genomic characteristics indicated that the two strains I/26-Cor1T and I/32A-Cor1 are representatives of the same species . The phylogenetic distance to any validly described taxon as indicated by 16S rRNA gene sequence similarities demonstrates that I/26-Cor1T and I/32A-Cor1 represent a novel species, for which the name Hymenobacter aerophilus sp . nov . is proposed, with the type strain I/26-Cor1T (= DSM 13606T = LMG 19657T) . I/32A-Cor1 (= DSM 13607 = LMG 19658) is another strain of the species Hymenobacter aerophilus . Since the taxonomic status of strain I/74-Cor2 within the genus Hymenobacter was not determined unambiguously, it is designated Hymenobacter sp . I/74-Cor2 (= DSM 13611 = LMG 19659).

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 383 - 90
Bartonella bovis Bermond et al . sp . nov . and Bartonella capreoli sp . nov., isolated from European ruminants; Bermond D et al.; Two novel species of Bartonella isolated from European ruminants are described . Bartonella capreoli sp . nov . was isolated from the blood of roe-deer (Capreolus capreolus) captured in Chize, France . The type strain is IBS 193T (= CIP 106691T = CCUG 43827T) . It is distinct from another European ruminant isolate that originated from a cow from a French herd of 430 dairy cattle . The latter isolate belongs to a novel species named Bartonella bovis Bermond et al . sp . nov . The type strain is strain 91-4T (= CIP 106692T = CCUG 43828T) . The two bacteria appeared as small, fastidious, aerobic, oxidase-negative, gram-negative rods . Their biochemical properties were similar to those of members of the genus Bartonella . The sequences of the 16S rRNA and citrate synthase genes obtained from the two type strains were highly related to sequences of the different Bartonella species . Hybridization values when testing type strains of recognized Bartonella species, obtained with the nuclease/trichloroacetic acid method, support the creation of two novel species.

J Agric Food Chem, 2002 Apr 10, 50(8), 2318 - 23
Bacterial removal of quinolizidine alkaloids and other carbon sources from a Lupinus albus aqueous extract; Santana FM et al.; Two Gram-negative bacterial strains capable of using lupanine, the predominant quinolizidine alkaloid in Lupinus albus, as a sole carbon source were isolated from soil in which L . albus and L . luteus had been grown {Santana, F . M . et al . J . Ind . Microbiol . 1996, 17, 110-115} . In the present study, we present results suggesting that these isolates are of potential interest for removing lupanine and other quinolizidine alkaloids (QA) from the effluent resulting from the wet processing of Lupinus seeds, at temperatures within the range 20-34 degrees C . Growth in L . albus aqueous extract was diauxic, with a first period of rapid growth leading to the simultaneous consumption of a significant part of the initial concentration of QA (3 g L(-1), being 2 g L(-1) lupanine) and amino acids (1.5 g L(-1)) . This period was followed by a second period of slower growth corresponding to the subsequent partial utilization (25%) of the carbohydrates (initial concentration of 20 g L(-1)) together with further removal of QA and amino acids . Despite the differences detected in the susceptibility of the two strains to lupanine toxicity, in particular at supraoptimal temperatures, and in the efficiency of lupanine catabolism, their performance on L . albus extract did not vary significantly.

Urology . 2002 Apr;59(4):601.
Staghorn calculus endotoxin expression in sepsis; McAleer IM et al.; Staghorn calculi are infrequent and generally are infected stones . Struvite or apatite calculi are embedded with gram-negative bacteria, which can produce endotoxin . Sepsis syndrome may occur after surgical therapy or endoscopic manipulation of infected or staghorn calculi . Sepsis, which can occur despite perioperative antibiotic use, may be due to bacteremia or endotoxemia . We present a child with an infected staghorn calculus who developed overwhelming sepsis and died after percutaneous stone manipulation . Endotoxin assay of stone fragments demonstrated an extremely high level of endotoxin despite low colony bacterial culture growth . This is the first reported case in which endotoxin was demonstrated in stone fragments from a child who died of severe sepsis syndrome after percutaneous staghorn stone manipulation.

Curr Infect Dis Rep, 2002 Apr, 4(2), 112 - 117
Mechanisms of Emerging Diarrheagenic Escherichia coli Infection; Khan MA et al.; Diarrheagenic Escherichia coli organisms are major causes of morbidity and mortality worldwide . Although most strains of E . coli are harmless commensals, a few types have emerged that are capable of disrupting the normal physiology of the human gut, producing illness ranging from watery diarrhea to fatal hemorrhagic colitis . Diarrheagenic E . coli cause infection by a variety of complex mechanisms, some of which are incompletely understood . These include adherence, elaboration of toxigenic mediators, invasion of the intestinal mucosa, and transportation of bacterial proteins into the host cells . Specific components of the host-microbial interaction that cause damage have been identified, increasing our understanding of the mechanisms of diarrhea . This article reviews some of the recent findings about the pathogenesis and infectious processes involved in three emerging pathotypes of this fascinating gram-negative bacterium.

Proteomics, 2002 Mar, 2(3), 313 - 24
Immunoproteomics of Helicobacter pylori infection and relation to gastric disease; Haas G et al.; The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer . A systematic, proteome based approach was chosen to detect candidate antigens of H . pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease . Sera from patients with active H . pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis . Overall, 310 antigenic protein species were recognized by H . pylori positive sera representing about 17% of all spots separated . Out of the 32 antigens most frequently recognized by H . pylori positive sera, nine were newly identified and 23 were confirmed from other studies . Three newly identified antigens which belong to the 150 most abundant protein species of H . pylori, were specifically recognized by H . pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522) . Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations . The data from these immunoproteomic analyses are added to our public database . This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.

Environ Sci Technol, 2002 Mar 1, 36(5), 891 - 900
Relative dominance of physical versus chemical effects on the transport of adhesion-deficient bacteria in intact cores from South Oyster, Virginia; Dong H et al.; Bacterial transport experiments were conducted using intact sediment cores collected from sites on the Delmarva Peninsula near South Oyster, VA, to delineate the relative importance of physical and chemical heterogeneity in controlling transport of an adhesion-deficient bacterial strain . Electron microscopy revealed that the sediments consisted of quartz and feldspar with a variable amount of clay and iron and aluminum hydroxide coatings on the grains . A nonmotile, gram-negative indigenous groundwater strain, designated as Comamonas sp . DA001, was injected into the cores along with a conservative tracer bromide (Br) . DA001 cells were 1.2 x 0.6 microm in size with a hydrophilic surface and a slightly negative surface charge . Bacterial breakthrough preceded that of Br . This differential advection phenomenon can be accounted for by reduction of the effective porosity for the bacteria relative to Br . The distribution of cells remaining in the core as determined by scintillation counting and phosphor imaging techniques was variable, ranging from nearly uniform concentrations throughout the core to exponentially decreasing concentrations with distance from the point of injection . The fraction of bacterial retention in the core was positively correlated with the abundance of the metal hydroxides and negatively correlated with grain size . Because grain size was inversely correlated with the abundance of the metal hydroxide coatings, it was necessary to separate the effects of grain size and mineralogy . The fraction of the bacterial retention accounting for the effect of grain size, the collision efficiency, exhibited no correlation with the abundance of the metal hydroxides, indicating that the bacterial retention was primarily controlled by grain size . Reasons for the lack of influence of mineralogy on bacterial transport include (i) the slightly negatively charged bacterial surfaces; (ii) an insufficient heterogeneity of sediment surface properties; and (iii) the masking of the positive charge of the metal hydroxide surfaces by adsorbed organic carbon (up to 1180 ppm) . This study demonstrates that the laboratory-based bacterial transport experiments are effective in delineating physical versus chemical controlling factors and provide an important link to field-based bacterial transport studies.

Appl Environ Microbiol, 2002 Apr, 68(4), 1548 - 55
Analysis of bacteria contaminating ultrapure water in industrial systems; Kulakov LA et al.; Bacterial populations inhabiting ultrapure water (UPW) systems were investigated . The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries . Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy . Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride {CTC} and DAPI {4',6'-diamidino-2-phenylindole} staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts . A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain . Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied . These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains . These bacteria constituted a significant part of the total number of isolated strains (>or=20%) . Two sets of primers specific to R . pickettii and Bradyrhizobium sp . were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples . Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp . and some P . saccharophilia strains isolated from UPW . The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.

Trends Microbiol, 2002 Apr, 10(4), 186 - 92
Port of entry--the type III secretion translocon; Buttner D et al.; Many Gram-negative plant and animal pathogenic bacteria use a specialized type III secretion system (TTSS) as a molecular syringe to inject effector proteins directly into the host cell . Protein translocation across the eukaryotic host cell membrane is presumably mediated by a bacterial translocon . The structure of this predicted transmembrane complex and the mechanism of transport are far from being understood . In bacterial pathogens of animals, several putative type III secretion translocon proteins (TTPs) have been identified . Interestingly, TTP sequences are not conserved among different bacterial species, however, there are structural similarities such as transmembrane segments and coiled-coil regions . Accumulating evidence suggests that TTPs are components of oligomeric protein channels that are inserted into the host cell membrane by the TTSS.

Biochem Cell Biol, 2002, 80(1), 41 - 8
Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide; Rossi P et al.; Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS) . We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane . This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations . The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+ . In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound . Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues . Ca2+ binding is characterized by an apparent dissociation constant of 6 microM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation . The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.

J Laparoendosc Adv Surg Tech A, 2002 Feb, 12(1), 69 - 72
An unusual case of Bouveret's syndrome; Harthun NL et al.; A 69-year-old man presented to the emergency department with a 12-hour history of severe abdominal pain . His medical history was significant for a small-bowel obstruction that resolved with conservative therapy 4 months prior to admission . In the distant past, a Billroth II gastric resection was performed for ulcer disease . He was hypothermic, and laboratory studies showed elevated serum liver and pancreatic enzymes . A CT scan of the abdomen demonstrated fat stranding and a small amount of free air in the area of the pancreas . Gram-negative rods subsequently grew from blood cultures . A presumptive diagnosis of necrotising pancreatitis was made, and supportive care was instituted . Follow-up CT scan performed several days later demonstrated a large filling defect in the stomach . Endoscopy showed this defect to be a giant gallstone, and the diagnosis of Bouveret's syndrome was made . The patient underwent laparotomy . A duodenal perforation in the posterior aspect of the fourth portion was identified . The perforation had been caused by chronic impaction of the giant stone . The stone was removed through the perforation, and the perforation was closed in multiple layers . Drainage of the retroperitoneum was effected through large catheters placed through the flank . The presentation, diagnostic evaluation, treatment, and complications of this condition are discussed.

Crit Care Med, 2002 Jan, 30(1), 38 - 43
No effect of preoperative selective gut decontamination on endotoxemia and cytokine activation during cardiopulmonary bypass: a randomized, placebo-controlled study; Bouter H et al.; BACKGROUND: Cardiopulmonary bypass predisposes the splanchnic region to inadequate perfusion and increases in gut permeability . Related to these changes, circulating endotoxin has been shown to rise during cardiac surgery, and may contribute to cytokine activation, high oxygen consumption, and fever ("postperfusion syndrome") . To a large extent, free endotoxin in the gut is a product of the proliferation of aerobic gram-negative bacteria and may be reduced by nonabsorbable antibiotics . OBJECTIVE: To evaluate the effect of preoperative selective gut decontamination (SGD) on the incidence of endotoxemia and cytokine activation in patients undergoing open heart surgery . DESIGN: Prospective, randomized, placebo-controlled double-blind trial . SETTING: Tertiary-care university teaching hospital . INTERVENTION: Preoperative administration for 5 to 7 days of oral nonabsorbable antibiotics (polymyxin B and neomycin) vs . placebo . The efficacy of SGD was assessed by culture of rectal swabs . PATIENTS: Forty-four patients (median age 65 yrs, 29 males) were included in a pilot study to establish the sampling points of perioperative measurements . Seventy-eight consecutive patients (median age 65 yrs, 55 males) were enrolled for the prospective study; of these, 51 were randomly allocated to take SGD (n = 24) or placebo (n = 27); 27 were included in a control group (no medication) . MEASUREMENTS AND RESULTS: SGD but not placebo effectively reduced the number of rectal swabs that grew aerobic gram-negative bacteria (27% vs . 93%, respectively; p < .001) . SGD did not affect the occurrence of perioperative endotoxemia, nor did it reduce the tumor necrosis factor-alpha, interleukin-10, or interleukin-6 concentrations (p > .20), as determined before surgery, upon aorta declamping, 30 mins into reperfusion, or 2 hrs after surgery . Also, SGD did not alter the incidence of postoperative fever or clinical outcome measures such as duration of artificial ventilation and intensive care unit and hospital stay . CONCLUSION: SGD effectively reduces the aerobic gram-negative bowel flora in cardiac surgery patients but fails to affect the incidence of perioperative endotoxemia and cytokine activation during cardiopulmonary bypass and the occurrence of a postperfusion syndrome.

Proc Natl Acad Sci U S A, 2002 Apr 2, 99(7), 4656 - 61 Epub 2002 Mar 19.
Bartonella-associated endothelial proliferation depends on inhibition of apoptosis; Kirby JE et al.; Bartonella is a Gram-negative pathogen that is unique among bacteria in being able to induce angioproliferative lesions . Cultured human endothelial cells have provided an in vitro system in which to study the basis of angioproliferation . Previous studies have attributed the organism's ability to induce angioproliferative lesions to direct mitotic stimulation of endothelial cells by these bacteria . Here we show that Bartonella inhibits apoptosis of endothelial cells in vitro, and that its ability to stimulate proliferation of endothelial cells depends to a large extent on its antiapoptotic activity . Bartonella suppresses both early and late events in apoptosis, namely caspase activation and DNA fragmentation, respectively . Its ability to inhibit death of endothelial cells after serum starvation can be recapitulated by media conditioned by bacteria, indicating that direct cell contact is not necessary . Among tested strains, the activity is produced only by Bartonella species that are significant human pathogens and are associated with angioproliferative lesions . We suggest that endothelial cells normally respond to infection by undergoing apoptosis and that Bartonella evolved the antiapoptotic activity to enhance survival of the host cells and therefore itself . We propose that Bartonella's antiapoptotic mechanism accounts at least in part for its ability to induce vascular proliferation in vivo.

Biochem J, 2002 Apr 1, 363(Pt 1), 105 - 15
Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50; Veith PD et al.; Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis . We have undertaken a proteomic study of the outer membrane of P . gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting . Proteins were identified by reference to the pre-release genomic sequence of P . gingivalis available from The Institute for Genomic Research . Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins . Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins . The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors . Several domains were found to be C-terminally truncated 16-57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine . This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp . Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing . The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13-42 kDa higher than that calculated from their gene sequences . The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.

Crit Care Med, 2002 Jan, 30(1), 23 - 31
Endogenous lipoproteins impact the response to endotoxin in humans; Harris HW et al.; OBJECTIVE: To determine whether endogenous lipoproteins can abrogate the host response to lipopolysaccharide (LPS) in vivo . DESIGN: Randomized, placebo-controlled study . SETTING: Urban public hospital with academic affiliation . SUBJECTS: Eighteen healthy, normolipidemic, normal weight volunteers, 21-35 yrs of age . INTERVENTIONS: Fasting and postprandial (hypertriglyceridemic) subjects were injected with endotoxin (LPS, Lot EC-5, 4 ng/kg = 20 endotoxin units/kg) as either a bolus or following preincubation of the LPS with autologous whole blood vs . saline . In addition, LPS-induced cytokine production was determined ex vivo to examine the capacity of fasting vs . hypertriglyceridemic whole blood to attenuate the effect of large, potentially lethal concentrations of LPS in humans . MEASUREMENTS: Vital signs were recorded and serial blood samples analyzed for changes in white blood cell count, cytokine, and stress hormone levels over 24 hrs . The distribution of lipoproteins in fasting and postprandial blood after preincubation was determined using 125I-LPS . MAIN RESULTS: Endogenous lipoproteins abrogated the host response to LPS in vivo, but only after preincubation with the LPS . Peak oral temperature (p < .05) and white blood cell count (p < .05), and plasma tumor necrosis factor (TNF)-alpha (p < .01) and adrenocorticotropic hormone levels (p < .03) were significantly reduced in volunteers injected with LPS preincubated with whole blood vs . LPS preincubated with saline . Approximately 80% of the LPS was bound to lipoproteins after preincubation with either fasting or hypertriglyceridemic blood . Thus, protection was associated with lipoprotein binding . In addition, hypertriglyceridemic but not fasting blood inhibited the ex vivo TNF-alpha response to large, highly toxic doses of LPS (p < .05) . Without the preincubation of lipoproteins with LPS, there was a trend for an exaggerated clinical and TNF-alpha response in the hypertriglyceridemic subjects . CONCLUSION: Preincubation of LPS with whole blood promotes lipoprotein-LPS binding and is associated with an attenuated response to this toxic macromolecule . Although the clinical relevance of these data requires further elucidation, our results continue to support a lipid-based therapeutic strategy to combat gram-negative sepsis.

Crit Care Med, 2002 Jan, 30(1), 171 - 81
Anti-ovine interleukin-1beta monoclonal antibody immunotherapy in an ovine model of gram-negative septic shock; Peake SL et al.; OBJECTIVE: To investigate the efficacy of an anti-ovine interleukin-1beta monoclonal antibody to ameliorate pathophysiological derangements and improve survival in an ovine model of gram-negative septic shock . DESIGN: Prospective, placebo-controlled, interventional study (24-hr study period) . SETTING: University hospital animal research laboratory . SUBJECTS: Ten awake, mature female sheep . INTERVENTIONS: Seven milligrams per kilogram of intravenous anti-ovine interleukin-1beta immunoglobin G1 monoclonal antibody (anti-interleukin-1beta group, n = 5) or equivalent amount of protein (5% human albumin; control group, n = 5) was infused over 1 hr (time-zero minus 1 hr to time-zero) and followed by an intravenous LD100 live Escherichia coli infusion (time-zero to time-zero plus 1 hr) . Normal saline, maintenance and boluses to maintain baseline filling pressures, and gentamicin, 3 mg/kg intravenous, at time-zero plus 2 and time-zero plus 13 hrs . MEASUREMENTS AND MAIN RESULTS: Hemodynamic and oxygen transport indexes as well as hematological, biochemical, cytokine (interleukin-1beta, tumor necrosis factor-alpha), and endotoxin measurements were performed at baseline (time-zero minus 1 hr), on completion of the monoclonal antibody/placebo (time-zero) and E . coli (time-zero plus 1 hr) infusions, and at multiple time points thereafter (time-zero plus 1.5 hrs to time-zero plus 24 hrs) . Baseline data were not different between the treatment groups . From time-zero plus 1.5 hrs onward, in the anti-interleukin-1beta group, there was a sustained increase in mean arterial pressure, decreased peripheral vasodilation, and an attenuated metabolic acidosis, relative to the control group (p < or = .01, repeated-measures analysis of variance) . Predicted percentage increases in mean arterial pressure and systemic vascular resistance index relative to the control group were 35% and 40%, respectively . Resuscitation fluid requirements were also decreased: anti-interleukin-1beta group, 4.1 +/- 2.9 mL x kg(-1) x hr(-1); control group, 10.6 +/- 1.8 mL x kg(-1) x hr(-1) (p < or = .01, Student's t-test) . Survival was not different (anti-interleukin-1beta group, 40%; control group, 0%; p > .01, log-rank test) . CONCLUSIONS: Adjunctive therapy with anti-ovine interleukin-1beta monoclonal antibody in ovine gram-negative septic shock was associated with improved hemodynamic performance . However, the beneficial effects were incomplete and survival was not significantly improved.

MMWR Morb Mortal Wkly Rep, 2002 Mar 8, 51(9), 181 - 4
Tularemia--United States, 1990-2000.
{Dog bite in a splenectomized patient}
Delanaye P, Dubois C, Mendes P, Bertholet M, Lambermont B.

Service de Nephrologie, CHU Sart TilmanWe present a case of Capnocytophaga canimorsus fulminant infection linked to a dog bite in a splenectomized patient . Capnocytophaga canimorsus is a gram-negative rod that typically causes septicaemia with disseminated intravascular coagulation in both immunocompromised and immunocompetent hosts . It is associated with high mortality . We also reviewed the literature and provide some recommendations on the management of bite wound as well as on both prevention and treatment of infection in asplenic state.

Am J Physiol Regul Integr Comp Physiol, 2002 Apr, 282(4), R979 - 84
Cytokine-mediated downregulation of vasopressin V(1A) receptors during acute endotoxemia in rats; Bucher M et al.; The reduced pressure response to vasopressin during acute sepsis has directed our interest to the regulation of vasopressin V(1A) receptors . Rats were injected with lipopolysaccharide for induction of experimental gram-negative sepsis . V(1A) receptor gene expression was downregulated in the liver, lung, kidney, and heart during endotoxemia . Inasmuch as the concentrations of proinflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were highly increased during sepsis, the influence of these cytokines on V(1A) receptor expression was investigated in primary cultures of hepatocytes and in the aortic vascular smooth muscle cell line A7r5 . V(1A) receptor expression was downregulated by the cytokines in a nitric oxide-independent manner . Blood pressure dose-response studies after injection of endotoxin showed a diminished responsiveness to the selective V(1) receptor agonist Phe(2),Ile(3),Orn(8)-vasopressin . Our data show that sepsis causes a downregulation of V(1A) receptors and suggest that this effect is likely mediated by proinflammatory cytokines . We propose that this downregulation of V(1A) receptors contributes to the attenuated responsiveness of blood pressure in response to vasopressin and, therefore, contributes to the circulatory failure in septic shock.

Infect Control Hosp Epidemiol, 2002 Feb, 23(2), 99 - 101
Central venous catheter-associated bloodstream infections in pediatric oncology home care; Shah SS et al.; Fifty-two pediatric oncology patients with central venous catheters (CVCs) who received home care services were studied . Gram-negative organisms were responsible for a greater proportion of CVC-associated bloodstream infections in pediatric oncology patients receiving home care than in hospitalized pediatric oncology patients.

Crit Care Med, 2002 Jan, 30(1 Supp), S64 - S73
Endotoxin tolerance: A review; West MA et al.; Endotoxin tolerance was initially described when it was observed that animals survived a lethal dose of bacterial endotoxin if they had been previously treated with a sublethal injection . In animal models, two phases of endotoxin tolerance are described, an early phase associated with altered cellular activation and a late phase associated with the development of specific antibodies against the polysaccharide side chain of Gram-negative organisms . Recently, there has been a tremendous resurgence of interest in the mechanisms responsible for altered responsiveness to bacterial endotoxin . Host immune cells, particularly macrophages and monocytes, that are exposed to endotoxin for 3 to 24 hrs are rendered "tolerant" and manifest a profoundly altered response when rechallenged with bacterial endotoxin or lipopolysaccharide . The "lipopolysaccharide-tolerant" phenotype is characterized by inhibition of lipopolysaccharide-stimulated tumor necrosis factor production, altered interleukin-1 and interleukin-6 release, enhanced cyclooxygenase-2 activation, inhibition of mitogen-activated protein kinase activation, and impaired nuclear factor-kappaB translocation . Human monocytes and macrophages can be induced to become tolerant, and there is increasing evidence that monocytic cells from patients with systemic inflammatory response syndrome and sepsis have many characteristics of endotoxin tolerance.

Int J Med Microbiol, 2002 Feb, 291(6-7), 463 - 7
The Dot/lcm transporter of Legionella pneumophila: a bacterial conductor of vesicle trafficking that orchestrates the establishment of a replicative organelle in eukaryotic hosts; Roy CR; Legionella pneumophila are gram negative bacteria that parasitize professional phagocytes . When ingested by their eukaryotic hosts, these bacteria have the unique ability to alter maturation of the endocytic vacuoles in which they reside initially and create an organelle that supports replication . This review will focus on several scientific breakthroughs made recently that shed light on the mechanisms by which L . pneumophila are able to establish a niche permissive for intracellular growth.

Crit Care Med, 2002 Feb, 30(2), 349 - 54
Evaluation of endotoxin release and cytokine production induced by antibiotics in patients with Gram-negative nosocomial pneumonia; Maskin B et al.; OBJECTIVE: To determine the plasma concentrations of lipopolysaccharide, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 in a homogeneous group of septic patients and to evaluate the effect of antibiotic treatment, imipenem or ceftazidime, on the release of lipopolysaccharide and cytokines . DESIGN: Prospective, randomized study . SETTING: Sixteen-bed multidisciplinary intensive care unit . PATIENTS: Twenty-four septic patients with documented Gram-negative nosocomial pneumonia . Controls were 20 patients admitted without sepsis and 20 healthy volunteers . INTERVENTIONS: Septic patients were randomized between imipenem and ceftazidime . Blood samples were collected before (0 hrs) and after (4 and 12 hrs) antibiotic treatment . Concentrations of lipopolysaccharide were measured by using the limulus assay, and cytokine concentrations were measured by enzyme-linked immunosorbent assay . Statistical analyses were performed by Kruskal-Wallis test, Mann-Whitney U test, and Student's t-test . MEASUREMENTS AND MAIN RESULTS: The mean age was 48.5 +/- 19.5 . The mean Acute Physiology and Chronic Health Evaluation II score was 18.4 +/- 4.5 . Overall mortality rate was 45.4% . All septic patients showed significant higher concentrations of lipopolysaccharide (p <.001), tumor necrosis factor-alpha (p <.04), and interleukin-6 (p <.001) than the controls, but interleukin-1 beta was never detected . We did not find statistically significant changes in lipopolysaccharide or cytokine plasma concentrations over time within any of the two arms of the study (ceftazidime vs . imipenem) . There were no statistically significant differences in lipopolysaccharide and interleukin-6 plasma concentrations between the two antibiotic treatments . Although tumor necrosis factor-alpha plasma concentrations were significantly higher in the group treated with ceftazidime compared with the group treated with imipenem at the baseline and 4 hrs later, these differences were not statistically significant after 12 hrs of initiation of both treatments . CONCLUSIONS: Patients with Gram-negative nosocomial pneumonia have high plasma concentrations of lipopolysaccharide, interleukin-6, and tumor necrosis factor-alpha, but the antibiotic therapy evaluated did not significantly modify these concentrations.

J Biol Chem, 2002 May 24, 277(21), 18281 - 90 Epub 2002 Mar 11.
Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases; Sweet CR et al.; Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes . It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response . Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions . These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA) . To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli . In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates . Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E . coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . In contrast, LpxA from B . parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP . This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A.

Odontostomatol Trop, 2001 Dec, 24(96), 26 - 9
{Noma and HIV infection: apropos of a case at the National Hospital Center in Bobo-Dioulasso (Burkina Faso)}; Ki-Zerbo GA et al.; Noma (Cancrum oris) is a gangrenous stomatitis arising from a periodontal infection and leading to severe soft tissue and bone destruction . The pathology involves numerous factors including local thrombosis, vascularitis, necrotizing gingivitis, immunodeficiency, gram negative and anaerobic infection . It is usually a disease of infants and malnourished children in tropical areas often occurring after a debilitating disease like measles . Recently, cases have been reported in adults especially elderly patients or during immunodeficiency states . Reconstructive surgery is often necessary to deal with destruction and sequel but is rarely accessible in developing countries . We report one case of noma (cancrum oris) in an HIV seropositive patient at the National Hospital in Bobo-Dioulasso . The noma was inaugural of AIDS in a 40 years old labourer coming back from Ivory Coast and no major opportunistic infection was associated . The course was fulminant leading to extensive facial gangrene with recurrent bacterial infections . The disease was fatal in this depressive, malnourished and diarrhoeic patient despite local surgical treatment, prolonged antibiotherapy and supportive care . Pathogenic mechanisms, management and preventive issues are discussed.

Jpn J Pharmacol, 2001 Nov, 87(3), 195 - 201
Deterioration of spatial learning performances in lipopolysaccharide-treated mice; Arai K et al.; It is well demonstrated that acute or chronic stress leads to reduction of learning ability . Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces profound physiological and behavioral changes, including fever, decrease in food motivation, and decrease in social behavior . These changes might be interpreted as an acute stress reaction to the LPS . In the present study, therefore, we investigated the effects of LPS (400-800 microg/kg, i.p.) on spatial learning performances using C57BL/6J male mice . In the Morris water-maze task, spatial learning performances were examined in six trials of training for two consecutive days . LPS-treated mice took a longer time to reach the hidden platform than control mice (F(1,60)=4.80801, P<0.05 at 600 microg/kg) . In addition, injection of LPS decreased the percent of correct choices in the Y-maze test (P<0.05 at 800 microg/kg) . LPS, however, did not alter the body weight, grip tone, motor activity or swimming speed . Taken together, these results indicate that LPS treatment specifically impaired spatial learning performances.

Ginekol Pol, 2001 Dec, 72(12A), 1334 - 9
{IL-6 and TNF-alpha levels in oviductal epithelial cells culture after escherichia coli LPS stimulation}; Palczynski B et al.; The aim of the study was to determine wether Gram-negative bacterial cell membrane lipopolysaccharides (endotoxine) can change the IL-6 and TNF-alpha cytokine concentration synthetized by fallopian tube endothelial cells . For the study 5 normal fallopian tubes from females at their reproductive age who underwent total hysterectomy due to uterine myoma were used . The fallopian tubes specimens (endothelial tissue) 2 mm2 fixed in 0.5 ml DMEM/HAM F-12 (GibcoBRL) solution with 15% FCS, Gentamycin, Fungizone, ITS (GibcoBRL) were incubated at 37 degrees C with 5% CO2 . The explants were stimulated by Escherichia coli lipopolysaccharide (LPS) at ascending concentrations 1 ng/mL, 10 ng/mL and 100 ng LPS/mL incubation media . Tissue specimen incubated in a media without LPS were used for control test . IL-6 activity in the supernatants were determined by Van Sinc method, TNF-alpha activity were determined against WEHI-164.13 . cells according to Espevik and Nisser-Mayer . The presence of TNF-alpha and IL-6 were confirmed in all the supernatants of the incubated fallopian tubes explants . The LPS stimulation caused a concentration increase in both cytokines . The maximum cytokine concentration level was observed in the incubation stimulated at 1 and 10 ng LPS/mL incubation media . The use of the highest LPS concentration retarded the cytokine production . CONCLUSION: The TNF-alpha and IL-6 cytokines dose-dependent production is caused by the fallopian tubes LPS stimulation.

J Clin Microbiol, 2002 Mar, 40(3), 1085 - 7
Biochemical and susceptibility tests useful for identification of nonfermenting gram-negative rods; Laffineur K et al.; Six hundred nineteen strains of nonfermenting gram-negative rods were tested for alkaline phosphatase, benzyl-arginine arylamidase, pyrrolidonyl arylamidase, ethylene glycol acidification, and susceptibility to desferrioxamine and colistin . The results were highly discriminant . Therefore, the proposed tests may be helpful for the identification of this group of organisms.

Am J Physiol Lung Cell Mol Physiol, 2002 Apr, 282(4), L796 - 802
Time-dependent reversal of sepsis-induced PMN uptake and lung vascular injury by expression of CD18 antagonist; Xu N et al.; We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice . Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter . We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression . Expression of both was evident at 1 h after E . coli challenge, peaked at 3-6 h, and returned to basal level within 48 h . We observed that increases in PMN uptake and transalveolar PMN migration induced by E . coli challenge were reversed in a time-dependent manner following NIF expression in mice . NIF expression also prevented the progression of lung vascular injury and edema formation following E . coli challenge . Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis . The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E . coli challenge.

Extremophiles, 2002 Feb, 6(1), 33 - 7
Marinospirillum alkaliphilum sp . nov., a new alkaliphilic helical bacterium from Haoji soda lake in Inner Mongolia Autonomous Region of China; Zhang W et al.; A new helical, alkaliphilic, gram-negative, chemoorganotrophic bacterium designated strain Z4T was isolated from Haoji soda lake in Inner Mongolia Autonomous Region, China . The isolate grows at salinities between 0.2% and 5.0% (w/v) NaCl and pH range 7.0-11.0, with an optimum at 2.0% (w/v) NaCl and pH 9.5 . Its growth temperature ranges from 8 degrees to 49 degrees C with an optimum at 37 degrees C . The G+C content of the DNA is 46.8 mol% . The major isoprenoid quinone is ubiquinone 8 (Q-8) . Phylogenetic analyses based on 16S rDNA sequence comparison indicates that strain Z4T is a member of the genus Marinospirillum . Phenotypic features and DNA-DNA homology of less than 20% with the described species of Marinospirillum support the view that strain Z4T represents a new species of the genus Marinospirillum . Strain Z4T (= AS 1.2746) is proposed as the type strain of a new species, named Marinospirillum alkaliphilum sp . nov.

J Pediatr Surg, 2002 Mar, 37(3), 390 - 4
Intestinal permeability and bacterial translocation are uncoupled after small bowel resection; O'Brien DP et al.; BACKGROUND/PURPOSE: Gut barrier failure and bacterial translocation have been proposed to cause infection and sepsis in patients with the short bowel syndrome . This study tested the hypothesis that permeability is increased in the adapting remnant ileum after massive small bowel resection (SBR) . METHODS: Male ICR mice underwent a 50% proximal SBR or sham operation . At 3, 7, and 14 days, the ileum was mounted in an Ussing chamber . Mucosal-to-serosal flux of low (dextran) and high (horseradish peroxidase; HRP) molecular weight markers was determined . Additionally, bacterial translocation was measured by culturing blood, mesenteric lymph nodes, liver, and spleen at 3 and 14 days after SBR or sham operation . RESULTS: Permeability to dextran was reduced immediately after SBR but was no different at later time-points . HRP permeability was no different at any time-point . Translocation of Gram-negative bacteria to the mesenteric lymph nodes and liver was more frequent in the SBR animals 3 and 14 days postoperatively . CONCLUSIONS: Intestinal permeability to macromolecules is not increased after massive SBR, but the rate of translocation to the mesenteric lymph nodes and liver is elevated . This suggests that the mechanism for bacterial translocation after SBR does not involve alterations in gut permeability .

Zhonghua Shao Shang Za Zhi, 2001 Apr, 17(2), 102 - 4
{A study on bactericidal/permeability increasing protein (BPI) as a natural inhibitor of endotoxin}; Zhou H; OBJECTIVE: To investigate the existential status of BPI as a natural defensive factor in human polymorphonuclear leukocytes (PMNL) . METHODS: The abilities of porcine BPI to combine endotoxin (lipopolysaccharide, LPS) and inhibit the release of TNFalpha from hepatic Kupffer's cells were examined . And the expression of BPI in PMNL after stimulation by LPS was determined by flow cytometry . RESULTS: The ability of porcine BPI to combine LPS was increased along with the increment of BPI concentration . The effects of the BPI on the inhibition of the release of TNFalpha from hepatic Kupffer's cells were dose-dependant . The expression of BPI in PMNL was enhanced obviously within 30 mins of LPS stimulation . But there was no immediate release of BPI into blood . CONCLUSION: BPI possessed potential power of neutralizing LPS . It might be beneficial to supplement exogenous BPI in case of Gram negative bacterial infection.

Arch Intern Med, 2002 Mar 11, 162(5), 594 - 9
Association between Chlamydia pneumoniae antibodies and intimal calcification in femoral arteries of nondiabetic patients; Lehto S et al.; BACKGROUND: Chlamydia pneumoniae, a gram-negative bacterium, has been suggested to be a risk factor for atherosclerosis . Calcium is a well-known component of atherosclerotic plaques, but it is uncertain whether infectious agents play a role in the calcification process of the arteries . PATIENTS: To address this issue we investigated the association of Chlamydia antibodies with intimal arterial calcification as assessed by soft tissue radiograms from the thigh region of 1373 nondiabetic Finnish individuals aged 45 to 64 years . RESULTS: At baseline, radiologically detectable intimal calcification in femoral arteries was found in 172 (27%) of 638 men and 43 (6%) of 735 women (P<.001) . The presence of intimal artery calcifications was strongly related to conventional atherosclerotic risk factors and to Chlamydia antibodies . In Cox regression analysis, association of Chlamydia antibodies with intimal artery calcification persisted after extensive adjustment for other cardiovascular risk factors (P =.04) . A dose-response relationship was observed between Chlamydia antibodies and intimal femoral artery calcification (P =.006) . The presence of intimal artery calcification was strongly associated with an increased risk of future coronary heart disease mortality (P<.001) . CONCLUSION: Chlamydia antibodies are strongly associated with intimal calcification of the femoral arteries.

Rinsho Byori, 2002 Jan, 50(1), 30 - 9
{High-sensitivity C-reactive protein (CRP) assay--a novel method for assessment of risk ratios for atherosclerotic vascular diseases}; Takahashi H; CRP has long been used as a sensitive marker for infectious diseases . Since its serum concentration elevates more than 10 mg/dl with gram-negative bacterial infections, the sensitivity can be enough to be around 0.3-0.6 mg/dl for the diagnosis . However, the sensitivity should be higher in the early diagnosis of infections in new-borne babies . In addition, recently, it was suggested that atherosclerotic lesions are a kind of vasculitis, and the information could be transmitted via production of inflammatory cytokines and acute phase proteins . In fact, serum CRP and serum amyloid protein A(SAA) levels are elevated even in patients with coronary atherosclerosis without acute coronary syndrome(ACS) . However, the level was much lower than the cut-off for diagnosis of bacterial infection . Therefore, the high-sensitive assay method has been applied . As the result, high-sensitivity(hs) CRP assay was found to be one of the most sensitive markers for prediction of future ACS in USA . Combination of hsCRP and atherogenic index such as total cholesterol/HDL cholesterol or LDL cholesterol/HDL cholesterol ratio is more useful . Similarly, it was found that hsCRP could predict the future prevalence of ACS even in Japan . It may be true because production of CRP is independent upon the genetic backgrounds . Early prevention of ACS by the measurement of hsCRP is calculated to be economic even if we measured hsCRF often in subjects without symptoms, because medical cost for treatment of acute myocardial infarction is enormous . In patients with high risk for coronary heart diseases, hsCRP-guided therapy is possible by using aspirin, stains, and antibiotics for prevention of ACS.

Trends Microbiol, 2002 Mar, 10(3), 147 - 52
Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions; Lara-Tejero M et al.; The coevolution of bacterial pathogens and their hosts has contributed to the development of very complex and sophisticated functional pathogen--host interfaces . Thus, well-adapted pathogens have evolved a variety of strategies to manipulate host cell functions precisely . For example, a group of unrelated Gram-negative pathogenic bacteria have evolved a toxin, known as cytolethal distending toxin (CDT), that has the ability to control cell cycle progression in eukaryotic cells . Recent studies have identified CdtB as the active subunit of the CDT holotoxin . Through its nuclease activity, CdtB causes limited DNA damage, thereby triggering the DNA-damage response that ultimately results in the observed arrest of the cell cycle . In addition, it has been established that CDT is a tripartite AB toxin in which CdtB is the active 'A' subunit and CdtA and CdtC constitute the heterodimeric 'B' subunit required for the delivery of CdtB into the target cell . The mechanism of action of CDT suggests that the infliction of limited damage could be a strategy used by pathogenic bacteria to modulate host cell functions.

Mol Genet Genomics, 2002 Feb, 266(6), 973 - 8 Epub 2001 Dec 01.
mRNA stability and the secretion signal of HrpA, a pilin secreted by the type III system in Pseudomonas syringae; Hienonen E et al.; Gram-negative bacteria that are pathogenic for animals or plants utilise a specialised Type III secretion system to inject effector proteins into their eukaryotic target cells . The basis for selection of the proteins to be translocated via type III systems is still enigmatic . No clearly defined consensus amino acid sequence that could serve as a specific secretion signal has been identified, and the hypothesis that an mRNA secondary structure acts as the signal has several shortcomings . We have localised a secretion signal that is sufficient to ensure the secretion of the pilin HrpA, a substrate and an indispensable extracellular component of the type III secretion machinery of Pseudomonas syringae pv . tomato DC3000, to the first 15 codons . Transcription of hrpA starts at a single site 42 bp upstream of the first codon . Gene swapping experiments revealed that altering the continuity of the 5' non-translated leader with the region including the secretion signal radically decreased accumulation of the hrpA transcript . These results indicate that an mRNA secondary structure, possibly formed in this region, is important for efficient expression of the gene . The proposed secondary structure is not, however, indispensable for the secretion of HrpA and it does not couple secretion and translation.

Am J Ther, 1996 Aug, 3(8), 586 - 596
New Therapeutic Approaches to Peptic Ulcer Disease: The Role of Helicobacter Pylori; Schlager SI et al.; One of the most common bacterial infections of human involves Helicobacter pylori, a spiral, gram-negative bacterium that is now thought to be a dominant factor in the development of peptic ulcer disease and may be significant in causing certain forms of gastric cancer . Almost 100% of patients with duodenal ulcer and 70 to 90% affected with gastric ulcer are infected with H . pylori . In order to achieve cure of H . pylori--induced ulcer disease, it is necesary to eradicate the bacterial infection . Mere suppression or clearance infection without eradication is associated with a >80% recurrence of the ulcer . The epidemiology, microbiology, and pathogenesis of H . pylori infections are reviewed . Diagnostic methods and optimal treatment strategies for H . pylori infections are examined . The most current diagnostic and treatment algorithms for peptic ulcer disease are discussed critically, and future directions for drug development aimed at eradication of H . pylori infection are considered.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2002 Feb, 93(2), 179 - 83
Fusobacterium nucleatum in endodontic flare-ups; Chavez de Paz Villanueva LE; OBJECTIVE: The extent to which Fusobacterium nucleatum is recovered from root canals of teeth that present with an interappointment flare-up following endodontic instrumentation was investigated . STUDY DESIGN: Included in the study were 28 patients that sought emergency treatment after initiation of root canal therapy . Only non-painful teeth that had been treated because of a necrotic pulp and periapical inflammatory lesion were studied . Root canal samples for bacterial analysis were taken, transported to a bacteriological laboratory, and processed for a semiquantitative assessment of bacterial isolates . Bacterial findings were correlated with self-assessed pain intensity as recorded by means of a Visual Analogue Scale . Clinical presentation of swelling and presence of exudate in the treated root canals were also linked . RESULTS: Bacteria were recovered from all teeth examined . Gram-negative anaerobic coccoid rods (Prevotella species and Porphyromonas species) were frequent isolates . All teeth in patients who were reported to be in severe pain (Visual Analogue Scale > or = 6) displayed F nucleatum . Nine out of 10 of these teeth also had swelling and exudate in the root canals . Samples from the remaining patients that had teeth with less pain score showed a variable bacterial recovery . None of these teeth displayed F nucleatum . CONCLUSION: F nucleatum appears to be associated with the development of the most severe forms of interappointment endodontic flare-ups.

Blood, 2002 Mar 1, 99(5), 1699 - 705
Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide; Nagai Y et al.; RP105 is a B-cell surface molecule that has been recently assigned as CD180 . RP105 ligation with an antibody induces B-cell activation in humans and mice, leading to proliferation and up-regulation of a costimulatory molecule, B7.2/CD86 . RP105 is associated with an extracellular molecule, MD-1 . RP105/MD-1 has structural similarity to Toll-like receptor 4 (TLR4)/MD-2 . TLR4 signals a membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS) . MD-2 is indispensable for TLR4-dependent LPS responses because cells expressing TLR4/MD-2, but not TLR4 alone, respond to LPS . RP105 also has a role in LPS responses because B cells lacking RP105 show hyporesponsiveness to LPS . Little is known, however, regarding whether MD-1 is important for RP105-dependent LPS responses, as MD-2 is for TLR4 . To address the issue, we developed mice lacking MD-1 and generated monoclonal antibodies (mAbs) to the protein . MD-1-null mice showed impairment in LPS-induced B-cell proliferation, antibody production, and B7.2/CD86 up-regulation . These phenotypes are similar to those of RP105-null mice . The similarity was attributed to the absence of cell surface RP105 on MD-1-null B cells . MD-1 is indispensable for cell surface expression of RP105 . A role for MD-1 in LPS responses was further studied with anti-mouse MD-1 mAbs . In contrast to highly mitogenic anti-RP105 mAbs, the mAbs to MD-1 were not mitogenic but antagonistic on LPS-induced B-cell proliferation and on B7.2 up-regulation . Collectively, MD-1 is important for RP105 with respect to B-cell surface expression and LPS recognition and signaling.

Thromb Haemost, 2002 Jan, 87(1), 155 - 62
Mechanism of resveratrol-mediated suppression of tissue factor gene expression; Pendurthi UR et al.; Tissue factor (TF) is a cell surface receptor for factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade . Inappropriate in vivo expression of TF in vascular cells has been shown to be responsible for thrombotic disorders associated with a variety of pathological conditions, including gram-negative sepsis, cancer and atherosclerosis . A number of epidemiological studies suggest that moderate consumption of red wine provides protective effects against coronary heart disease mortality . Recently, we have shown that resveratrol, a polyphenolic compound found in wine, inhibited the induction of TF expression in endothelial cells and mononuclear cells (Pendurthi UR, Williams JT, Rao LVM . Arterioscler Thromb Vasc Biol 1999: 19: 419-426) . In the present study, we examined the mechanism by which resveratrol inhibits the expression of TF in monocytes by using a monocytic cell line, THP-1, as a model cell . Northern blot analysis, gel mobility shift assays and transfection studies with various TF promoter constructs, as well as other transcription regulatory constructs, were used to elucidate the inhibitory mechanism of resveratrol . The data show that resveratrol inhibited lipopolysaccharide (LPS)-induced expression of TF in human monocytes and monocytic cell line, THP-1 in a dose dependent manner . Resveratrol did not significantly alter the binding of various transcription factors involved in TF gene expression to DNA . However, resveratrol suppressed the transcription of cloned human TF promoter . Further experiments revealed that resveratrol reduced kappaB- but not AP-1-driven transcriptional activity . Additional experiments showed that resveratrol suppressed the phosphorylation of p65 and its transactivation . In summary, our results indicate that resveratrol does not inhibit the activation or translocation of NF-kappaB/Rel proteins but inhibits NF-kappaB/Rel-dependent transcription by impairing the transactivation potential of p65.

Dig Dis Sci, 2002 Feb, 47(2), 380 - 3
Helicobacter pylori seroprevalence in patients with autoimmune hepatitis; Durazzo M et al.; Autoimmune hepatitis is characterized by a continuing hepatocyte necrosis that usually progresses to liver cirrhosis . Autoimmunity is also a feature of chronic infection by Helicobacter pylori, a gram-negative bacterium involved in the pathogenesis of peptic ulcer and upper gastrointestinal bleeding, with both events frequently occurring in patients with chronic liver disease . A newly described pathogenetic mechanism for chronic hepatitis and hepatocellular carcinoma in the mouse is linked to Helicobacter spp . infection . A high prevalence of H . pylori infection was demonstrated in patients with viral-related cirrhosis but never studied in cases of autoimmune hepatitis . In a case-control study, we examined 31 consecutive patients (25 women and 6 men, age range 20-66, mean age 46 +/- 4.3 years) suffering from autoimmune hepatitis and 62 sex- and age-matched blood donors (50 women, 12 men, age range 20-65, mean age 46 +/- 5.4 years) resident in the same area . Antibodies to H . pylori were present in 20 of 31 (64.5%) autoimmune patients compared to 33 of 62 (53.2%) controls (P = 0.3, odds ratio 1.60, 95% CI 0.60-4.28) . The difference was not statistically significant either in female or male patients . In conclusion, the prevalence of H . pylori infection in patients and controls was similar in our study of patients with chronic autoimmune hepatitis.

Infect Immun, 2002 Mar, 70(3), 1640 - 4
Role of cholesterol and the ganglioside GM(1) in entry and short-term survival of Brucella suis in murine macrophages; Naroeni A et al.; Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals . These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells . Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells . We have previously shown that the maturation inhibition of the Brucella-containing phagosome appears to be restricted at the phagosomal membrane, but the precise molecular mechanisms and factors involved in this inhibition have yet to be identified . Interestingly, recent studies have revealed that caveolae or lipid rafts are implicated in the entry of some microorganisms into host cells and mediate an endocytic pathway avoiding fusion with lysosomes . In this study, we investigated the role of cholesterol and the ganglioside GM(1), two components of lipid rafts, in entry and short-term survival of Brucella suis in murine macrophages, by using cholesterol-sequestering (filipin and beta-methyl cyclodextrin) and GM(1)-binding (cholera toxin B) molecules . Our results suggest that lipid rafts may provide a portal for entry of Brucella into murine macrophages under nonopsonic conditions, thus allowing phagosome-lysosome fusion inhibition, and provide further evidence to support the idea that the phagosome maturation inhibition is restricted at the phagosomal membrane.

Prikl Biokhim Mikrobiol, 2002 Jan-Feb, 38(1), 29 - 34
{Development of biosensors for phenol determination from bacteria found in petroleum fields of West Siberia}; Makarenko AA et al.; Nine Gram-negative bacterial strains, selected from 300 strains isolated from soils of the West Siberian petroliferous basin and growing on oil and oil products, consume phenol as a single carbon and energy source . The strains were used for the development of a sensor bioreceptor . The most active 32-I strain was shown to bear a plasmid responsible for phenol degradation . The plasmid-free derivative of this strain, 32-I-1, did not grow on phenol . The possibility of creating a model biosensor for phenol based on the plasmid-containing 32-I strain is considered . The detection limit for phenol was 5 microM . The optimum conditions for the sensor operation are: pH 7.4, 35 degrees C, and operation time 30 h.

J Vet Med B Infect Dis Vet Public Health, 2001 Dec, 48(10), 771 - 8
Development of a PCR-based method for specific identification of genotypic markers of shiga toxin-producing Escherichia coli strains; Osek J et al.; A simple, rapid and specific PCR-based method for identification of shiga toxin-producing Escherichia coli (STEC) was developed . The procedure involves amplification of the E . coli-specific universal stress protein A (uspA) gene (uspa-PCR), with the primer pair described by other authors, which allows differentiation of E . coli (STEC and non-STEC) from other gram-negative bacteria followed by identification of the main genetic virulence traits of the uspA-positive isolates . For this purpose, two multiplex PCR assays, based on previously published primer sequences, were established . Assay 1 (mPCR-1) uses three primer pairs and detects the genes encoding O157 (rfb), enterohemolysin (ebly) and shiga toxin (stx), generating amplification products of 420, 534 and 230 bp, respectively . Assay 2 (mPCR-2) uses four primer pairs specific for rfb (E . coli O157), eaeA (intimin), stx1 and stx2 (shiga toxin 1 and 2, respectively), generating PCR amplicons of 420, 840, 348 and 584 bp, respectively . These two assays were validated by testing several E . coli reference strains and 202 previously characterized E . coli isolates originating from calves and from children, and 100% agreement with previous results was obtained . The method developed can be used for specific identification of STEC bacteria including those of the O157 serogroup.

Immunol Lett, 2002 Apr 1, 81(1), 71 - 5
Toll-like receptor 4 surface expression on human monocytes and B cells is modulated by IL-2 and IL-4; Mita Y et al.; Human Toll-like receptor 4 (TLR4) has recently been identified, and it has been shown to be the main protein involved in recognizing gram-negative bacteria . We examined the regulation of TLR4 surface expression in human peripheral blood monocytes and B cells by interleukin-2 (IL-2) and IL-4 . IL-2 up-regulated TLR4 surface expression on human peripheral blood monocytes, but did not change expression on human peripheral B cells . By contrast, IL-4 down-regulated TLR4 surface expression on human peripheral blood monocytes, but up-regulated TLR4 surface expression on human peripheral B cells . These results indicate that Th1 cytokine IL-2 enhances receptors involved in the response to gram-negative bacteria and that activation of cellular immunity may enhance defense against these pathogens through monocytes, but not B cells, whereas Th2 cytokine IL-4 modulates the receptor response to gram-negative bacteria and that activation of humoral immunity may enhance defense against these pathogens through B cells, but not monocytes.

Proteomics, 2002 Feb, 2(2), 164 - 86
Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2; Shaw AC et al.; Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria . The genome of C . trachomatis D comprises 894 open reading frames (ORFs) . In this study the global expression of genes in C . trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach . Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C . trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used . Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C . trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C . trachomatis D genome and the 7.5 kb C . trachomatis plasmid . Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins . Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance . The availability of the complete genome made it feasible to map and to identify proteins of C . trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

Am J Pathol, 2002 Feb, 160(2), 739 - 51
Helicobacter bilis infection accelerates and H . hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr1a-/-) mice; Maggio-Price L et al.; mdr1a-deficient mice lack P-glycoprotein and spontaneously develop colitis with age . Helicobacter spp . are gram-negative organisms that have been associated with colitis in certain mouse strains, but Helicobacter spp . have been excluded as contributing to the spontaneous colitis that develops in mdr1a-/- mice . We wished to determine whether infection with either H . bilis or H . hepaticus would accelerate the development of inflammatory bowel disease (IBD) in mdr1a-/- mice . We found that H . bilis infection induced diarrhea, weight loss, and IBD in mdr1a-/- mice within 6 to 17 weeks post-inoculation and before the expected onset of spontaneous IBD . Histopathology of H . bilis-induced IBD included crypt hyperplasia, inflammatory cell infiltrates, crypt abscesses, and obliteration of normal gut architecture . Reverse transcription-polymerase chain reaction and Taqman analysis from colonic tissue showed increased transcripts for interferon-gamma and interleukin-10 from H . bilis-infected colitic mdr1a-/- mice . Additionally, mesenteric lymph nodes had increased cellularity with expansion of CD4+ and CD8+ T cells and B cells and increased proliferation to soluble H . bilis antigens with elaboration of interferon-gamma, tumor necrosis factor-alpha and interleukin-10 . In contrast, H . hepaticus infection of mdr1a-/- mice did not accelerate disease but rather delayed the onset of spontaneous colitis which was milder in severity . mdr1a-/- mice infected with Helicobacter spp . may provide a useful tool to explore the pathogenesis of microbial-induced IBD in a model with a presumed epithelial cell "barrier" defect.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 263 - 71
Pseudoalteromonas maricaloris sp . nov., isolated from an Australian sponge, and reclassification of {Pseudoalteromonas aurantia} NCIMB 2033 as Pseudoalteromonas flavipulchra sp . nov; Ivanova EP et al.; A marine, gram-negative, aerobic bacterium that produced cytotoxic, lemon-yellow, chromopeptide pigments that inhibited the development of sea urchin eggs has been isolated from the Australian sponge Fascaplysinopsis reticulata Hentschel . The cells of the organism were rod-shaped with a single polar flagellum and they required NaCl for growth (0.5-10%) with optimum growth at 1-3% NaCl . The temperature for growth was 10-37 degrees C, with optimum growth at 25-30 degrees C . Growth occurred at pH values from 6.0 to 10.0, with optimum growth at pH 6.0-8.0 . Major phospholipids were phosphatidylethanolamine, phosphatidylglycerol and lyso-phosphatidylethanolamine . Of 26 fatty acids with 11-19 carbon atoms that were detected, 16:1omega7, 16:0, 17:1omega8 and 18:1omega7 were predominant . The DNA G+C content was 38.9 mol% . All of these phenotypic and chemotaxonomic characters place the organism in the genus Pseudoalteromonas (Gauthier et al, 1995) . These data are consistent with the phylogenetic analyses that confirmed that strain KMM 636T is a member of the Pseudoalteromonas cluster in the gamma-subclass of the Proteobacteria . DNA-DNA hybridization experiments revealed that the levels of relatedness between the DNA of the strain studied and DNAs of type strains of the species that clustered together (on the basis of 16S rDNA sequences) and {Pseudoalteromonas aurantia} NCIMB 2033 ranged from 19 to 35%, and that the DNA-DNA homology between {P . aurantia} NCIMB 2033 and other phylogenetically and/or phenotypically similar type strains ranged from 32 to 52% . According to the polyphasic evidence presented in this study, it is proposed that strain KMM 636T (= LMG 19692T = CIP 106859T) be classified as Pseudoalteromonas maricaloris sp . nov . and {P . aurantia} NCIMB 2033 be reclassified as Pseudoalteromonas flavipulchra NCIMB 2033T (= KMM 3630T = LMG 20361T) sp . nov.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 251 - 61
Methylocapsa acidiphila gen . nov., sp . nov., a novel methane-oxidizing and dinitrogen-fixing acidophilic bacterium from Sphagnum bog; Dedysh SN et al.; A novel genus and species, Methylocapsa acidiphila gen . nov., sp . nov., are proposed for a methane-oxidizing bacterium isolated from an acidic Sphagnum peat bog . This bacterium, designated strain B2T, represents aerobic, gram-negative, colourless, non-motile, curved coccoids that form conglomerates covered by an extracellular polysaccharide matrix . The cells use methane and methanol as sole sources of carbon and energy and utilize the serine pathway for carbon assimilation . Strain B2T is a moderately acidophilic organism with growth between pH 4.2 and 7.2 and at temperatures from 10 to 30 degrees C . The cells possess a well-developed system of intracytoplasmic membranes (ICM) packed in parallel on only one side of the cell membrane . This type of ICM structure represents a novel arrangement, which was termed type III . The resting cells are Azotobacter-type cysts . Strain B2T is capable of atmospheric nitrogen fixation; it possesses particulate methane monooxygenase and does not express soluble methane monooxygenase . The major phospholipid fatty acid is 18:1omega7c and the major phospholipids are phosphatidylglycerols . The G+C content of the DNA is 63.1 mol% . This bacterium belongs to the alpha-subclass of the Proteobacteria and is most closely related to the acidophilic methanotroph Methylocella palustris KT (97.3% 16S rDNA sequence similarity) . However, the DNA-DNA hybridization value between strain B2T and Methylocella palustris K(T) is only 7% . Thus, strain B2T is proposed to comprise a novel genus and species, Methylocapsa acidiphila gen . nov., sp . nov . Strain B2T (= DSM 13967T = NCIMB 13765T) is the type strain.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 195 - 205
Shewanella frigidimarina and Shewanella livingstonensis sp . nov . isolated from Antarctic coastal areas; Bozal N et al.; Three strains of psychrophilic bacteria isolated from Antarctic coastal marine environments were studied to determine their taxonomic position . These bacteria were gram-negative rods, facultatively anaerobic and motile by means of a single polar flagellum . None of the bacterial isolates had an Na+ requirement . Only one of the strains was capable of producing H2S from thiosulfate . The DNA base content of these bacteria was 41-42 mol % G+C . DNA-DNA hybridization experiments showed that the isolates formed two related groups that exhibited about 70 and 24% DNA-DNA homology, respectively, with the type strain of Shewanella frigidimarina . The fatty acid profiles of the bacterial isolates were similar to the profiles of other Shewanella species . All the strains contained both ubiquinones and menaquinones, like Shewanella species . Methylmenaquinones were also found . 16S rRNA gene analysis confirmed that isolated strains belonged to the genus Shewanella and were phylogenetically related to the newly identified Shewanella frigidimarina . The results of the polyphasic taxonomic study assigned the three isolates to Shewanella and two of them specifically to Shewanella frigidimarina . The name Shewanella livingstonensis sp . nov . (type strain LMG 19866T) is proposed for the third organism.

Life Sci, 2001 Dec 28, 70(6), 615 - 28
The administration of lipopolysaccharide, in vivo, induces alteration in L-leucine intestinal absorption; Abad B et al.; The objective of the present study was to determine the alterations in L-leucine intestinal uptake by intravenous administration of Lipopolysaccharide (LPS), which is a constituent of gram negative bacterial, causative agent of sepsis . The amino acid absorption in LPS treated rabbits was reduced compared to the control animals . The LPS effect on the amino acid uptake was due to an inhibition of the Na+-dependent system of transport, through both reduction of the apparent capacity transport (Vmax) and diminution of the Na+/K-ATPase activity . The results have also shown that the LPS decreases the mucosal to serosal transepithelial flux and the transport across brush border membrane vesicles of L-leucine . The study of possible intracellular mechanisms implicated in the LPS effect, showed that the second messengers calcium, protein kinase C and c-AMP did not play any role in this effect . However, the absence of ion chloride in the incubation medium removes the LPS inhibition and the intracellular tissue water was affected by the LPS treatment . Therefore, the inhibition in the L-leucine intestinal absorption, by intravenous administration of LPS, could be mainly produced by the secretagogue action of this endotoxin on the gut.

J Endod, 2002 Feb, 28(2), 86 - 9
Comparison of the effectiveness of bacterial culture, 16S rDNA directed polymerase chain reaction, and checkerboard DNA-dNA hybridization for detection of Fusobacterium nucleatum in endodontic infections; Moraes SR et al.; Fusobacterium nucleatum is a Gram-negative, non-spore-forming, nonmotile, obligatory anaerobic rod that is normally isolated from the oral cavity . Epidemiological studies have shown that this species is one of the most prevalent in primary root canal infections . The purpose of this study was to compare the effectiveness of bacteriological culture, 16S rDNA directed polymerase chain reaction and checkerboard DNA-DNA hybridization for detection of F . nucleatum strains in infected teeth associated with periradicular lesions . Thirteen single-root teeth from adult patients, all having carious lesions, necrotic pulps, and radiographic evidence of periradicular bone loss were included in this study . Combining all methods, the results indicated that F . nucleatum was present in approximately 31% (4 of 13) of the specimens . Incidence of F . nucleatum in root canal infections, as evaluated in this study by polymerase chain reaction, culture, and DNA-DNA hybridization, was 15.4%, 15.4%, and 10.0%, respectively . Our data demonstrated that no method used herein could be considered superior for detecting F . nucleatum directly from clinical samples . However, the small number of samples examined and the low prevalence that was observed should be considered.

Rev Argent Microbiol, 2001 Oct-Dec, 33(4), 203 - 8
{Production and characterization of monoclonal antibodies directed agains the major outer membrane protein and lipopolysaccharide of Chlamydia trachomatis}; Cuffini C et al.; Two monoclonal antibodies (MnAb) directed against the lipopolysaccharide (LPS) and major outer membrane protein (MOMP) of Chlamydia trachomatis were produced for use in indirect immunofluorescence (IFI) . The specificity of the antibodies was determined by Dot-blot, Immunoblotting (IB) and IFI onto culture cells infected with C . trachomatis and IFI onto commercial swabs (MRL) . The MnAb 2D3 and 3C2 detected LPS and MOMP of C . trachomatis, respectively, by different methods . Neither MnAb showed cross-reactions when other gram-negative bacteria were used as antigens.

J Biol Chem, 2002 Apr 19, 277(16), 14194 - 205 Epub 2002 Feb 05.
A triple mutant of Escherichia coli lacking secondary acyl chains on lipid A; Vorachek-Warren MK et al.; All possible combinations of insertion mutations in the three genes encoding the acyl carrier protein-dependent late acyltransferases of lipid A biosynthesis, designated lpxL(htrB), lpxM(msbB), and lpxP, were generated in Escherichia coli K12 W3110 . Mutants defective in lpxM synthesize penta-acylated lipid A molecules and grow normally . Strains lacking lpxP fail to incorporate palmitoleate into their lipid A at 12 degrees C but make normal amounts of hexa-acylated lipid A and are viable . Although lpxL mutants and lpxL lpxM double mutants grow slowly on minimal medium at all temperatures, they do not grow on nutrient broth above 32 degrees C . Such mutants retain the ability to synthesize some penta- and hexa-acylated lipid A molecules because of limited induction of lpxP at 30 degrees C but not above 32 degrees C . MKV15, an E . coli lpxL lpxM lpxP triple mutant, likewise grows slowly on minimal medium at all temperatures but not on nutrient broth at any temperature . MKV15 synthesizes a lipid A molecule containing only the four primary (R)-3-hydroxymyristoyl chains . The outer membrane localization and content of lipid A are nearly normal in MKV15, as is the glycerophospholipid and membrane protein composition . However, the rate at which the tetra-acylated lipid A of MKV15 is exported to the outer membrane is reduced compared with wild type . The integrity of the outer membrane of MKV15 is compromised, as judged by antibiotic hypersensitivity, and MKV15 undergoes lysis following centrifugation . MKV15 may prove useful as a host strain for expressing late acyltransferase genes from other Gram-negative bacteria, facilitating the re-engineering of lipid A structure in living cells and the design of novel vaccines.

J Biol Chem, 2002 Apr 19, 277(16), 14274 - 80 Epub 2002 Feb 05.
Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in gram-negative sepsis and causes inflammation and death in mice; Hellman J et al.; Gram-negative bacterial sepsis commonly causes organ dysfunction and death in humans . Although circulating bacterial toxins trigger inflammation in sepsis, little is known about the composition of bacterial products released into the blood during sepsis or the contribution of various bacterial components to the pathogenesis of sepsis . We have shown that diverse Gram-negative bacteria release bacterial peptidoglycan-associated lipoprotein (PAL) into serum . The present studies explored release of PAL into the blood during sepsis and tested the hypothesis that PAL contributes to bacterial virulence and inflammation in Gram-negative sepsis . Released PAL was detected in the blood of 94% of mice following cecal ligation and puncture . Picomolar to nanomolar levels of PAL stimulated macrophages and splenocytes from lipopolysaccharide-hyporesponsive (C3H/HeJ) mice . Injection of PAL into C3H/HeJ mice stimulated production of serum cytokines and increased pulmonary and myocardial expression of inflammatory markers . PAL caused death in sensitized C3H/HeJ mice . Mutant Escherichia coli bacteria with reduced levels of PAL or truncated PAL were less virulent than wild-type bacteria, as indicated by higher survival rates and lower circulating levels of interleukin 6 and bacteria in a model of peritonitis in lipopolysaccharide-responsive mice . The studies suggest that PAL may be an important bacterial mediator of Gram-negative sepsis.

Zhonghua Wai Ke Za Zhi, 1999 May, 37(5), 278 - 81
{Monitoring of bacteria resistance in burn patients}; Zhang M et al.; OBJECTIVE: To monitor the bacteria resistance in burn patients . METHODS: Disk susceptibility tests were performed and interpreted according to the NCCLS criteria . Four kinds of the third generation cephalosporins extended-spectrum beta-lactamase produced by multi-resistant of P . aeruginosa and K . Pneumoniae strains were detected after 20 microg of sulbactam was added respectively, in contrast to no sulbactam . RESULTS: 227 strains were isolated from burn patients . 195 strains (86%) were gram-negative bacteria . Disk susceptibility showed various bacteria had high antibiotic resistance and multi-resistant rate . S . aureus was only susceptible to vancomycin, its resistant rate to imipenem was 19% . P . aeruginosa was only susceptible to polymyxin-B, its resistant rate to ceftazidime was 20% . However, after stop using ceftazidime two years, the susceptibility to gram-negative bacteria recovered . The resistant rate of ceftazidime to P . aeruginosa, E . coli, K . pneumoniae were decreased respectively . The resistance to quinolones was increased . The resistant rate of ciprofloxacin to P . aeruginosa, K . pneumoniae was increased respectively . After 20 microg sulbactam added to cephalosporins drug disks, the primary susceptibility of ceftazidime to P . aeruginosa and K . pneumoniae recovered, and the antibiotic was better than the other cephalosporins . CONCLUSIONS: It is important to monitor the bacteriology in burn patients at all time, and understand the changing pattern of bacterial flora, antibiotic susceptibility and bacterial strains spreading in burn ward . Extended-spectrum beta-lactamases is the cause of resistance . After sulbactam added to the third generation of cephalosporins, the beta-lactamases were inhibited, and the susceptibility of antibiotics to bacteria were increased and ceftazidime was prior to others.

J Clin Invest, 2002 Feb, 109(3), 419 - 25
The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta; Bannerman DD et al.; Activation of NF-kappa B by bacterial LPS promotes the upregulation of proinflammatory cytokines that contribute to the pathogenesis of Gram-negative septic shock . LPS activation of NF-kappa B is dependent upon the interaction of two death domain-containing (DD-containing) proteins, MyD88 and IL-1 receptor-associated kinase IRAK . Another DD-containing protein, Fas-associated death domain (FADD), also binds MyD88 through respective DD-DD interactions . Although FADD has been classically described as a proapoptotic signaling molecule, several reports have implicated a role for FADD in mediating NF-kappa B activation . In the present report, we investigated whether FADD could mediate LPS activation of NF-kappa B . Overexpression of FADD blocked LPS-induced NF-kappa B activation, whereas absence of FADD enhanced activation of NF-kappa B by LPS . Further, LPS-induced expression of two NF-kappa B-dependent gene products, IL-6 and KC, was enhanced in FADD(-/-) mouse embryo fibroblasts (MEFs) compared with wild-type . This increase in NF-kappa B activity correlated with enhanced I kappa B degradation . FADD(-/-) MEFs were also resistant to NF-kappa B activation induced by IL-1 beta . Finally, reconstitution of full-length FADD in the FADD(-/-) MEFs completely reversed the enhanced activation of NF-kappa B elicited by either LPS or IL-1 beta . Together, these data indicate that FADD negatively regulates LPS- and IL-1 beta-induced NF-kappa B activation and that this regulation occurs upstream of I kappa B degradation.

J Clin Microbiol, 2002 Feb, 40(2), 512 - 8
Multiserotype enzyme-linked immunosorbent assay as a diagnostic aid for periodontitis in large-scale studies; Pussinen PJ et al.; Periodontitis is a common chronic oral infection caused by gram-negative bacteria, including Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis . Periodontitis evokes inflammatory host response locally in the periodontium but also systemically . The systemic humoral antibody response against oral pathogens can conveniently be measured by an immunoassay . The aim of the study was to measure serum immunoglobulin G class antibodies against A . actinomycetemcomitans and P . gingivalis by an enzyme-linked immunosorbent assay (ELISA) in which mixtures of several serotypes of the pathogens were used as antigens to avoid biasing of the results in favor of a particular strain . For A . actinomycetemcomitans the antigen consisted of six strains representing serotypes a, b, c, d, and e and one nonserotypeable strain . In the P . gingivalis ELISA, antigens representing serotypes a, b, and c were used . Serum samples from 90 subjects, including 35 samples from patients with diagnosed periodontitis, 10 samples from periodontally healthy controls, and 45 samples from randomly selected apparently healthy volunteers (referred to as "healthy subjects"), were tested . For both pathogens the antibody levels (means +/- standard deviations) of the patients--xpressed as area under the dilution curve--were significantly higher than those for healthy controls or healthy subjects, with values for A . actinomycetemcomitans and P . gingivalis, respectively, as follows: patients, 22.60 +/- 9.94 mm(2) and 26.72 +/- 11.13 mm(2); healthy controls, 9.99 +/- 3.92 mm(2) and 6.90 +/- 3.38 mm(2); and healthy subjects, 16.85 +/- 6.67 mm(2) and 8.51 +/- 4.23 mm(2) . The serotype mixture ELISA is suitable for measuring antibodies against periodontal pathogens in large epidemiological studies in order to evaluate the role of periodontitis as a risk factor for other diseases.

Syst Appl Microbiol, 2001 Nov, 24(3), 358 - 61
Characterization of some strains from human clinical sources which resemble "Leptotrichia sanguinegens": description of Sneathia sanguinegens sp . nov., gen . nov; Collins MD et al.; Three strains of a gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods . Comparative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated "Leptotrichia sanguinegens" . Based on phylogenetic and phenotypic evidence, it is proposed that the isolates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen . nov., sp . nov . The type strain of Sneathia sanguinegens is CCUG 41628T.

Alcohol Clin Exp Res, 2002 Jan, 26(1), 74 - 82
A role for interleukin-10 in alcohol-induced liver sensitization to bacterial lipopolysaccharide; Hill DB et al.; BACKGROUND: Proinflammatory cytokines play an important role in alcohol-induced liver injury . The role of anti-inflammatory cytokines in the initiation and progression of alcoholic liver disease has received little attention . This study tested the hypothesis that an imbalance exists between pro- and anti-inflammatory cytokines in the liver during chronic exposure to alcohol . Alcohol exposure results in predominantly proinflammatory cytokine secretion and liver injury . METHODS: IL-10 knock-out and their C57BL/6J counterpart wild-type mice were fed alcohol in drinking water for 7 weeks . At the end of alcohol feeding, Gram-negative bacterial lipopolysaccharide (LPS) was administered, and the animals were killed after 3 and 8 hr . Liver histology, plasma alanine aminotransferase and aspartate aminotransferase activity, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-10 levels, and liver cytokine messenger RNA levels were measured . RESULTS: Alcohol feeding and LPS treatment did not change plasma enzyme activity levels in wild-type mice . In the IL-10 knock-out mice, LPS alone increased aspartate aminotransferase and alanine aminotransferase enzyme activity, and this was potentiated by alcohol . Alcohol induced liver steatosis in both wild-type and knock-out mice . LPS markedly enhanced the histological effects further, especially in the knock-out mice, with the emergence of focal necrosis, polymorphonuclear infiltration, and microabscesses in the liver . Plasma tumor necrosis factor-alpha and IL-1beta levels were not affected by alcohol alone . Proinflammatory cytokine levels were increased by LPS and further enhanced by alcohol treatment, particularly in the IL-10 knock-out mice . IL-10 plasma levels in the wild-type animals were down-regulated by alcohol . Changes in liver cytokine messenger RNA paralleled those seen in plasma cytokine levels . CONCLUSIONS: Alcohol-induced liver sensitization to LPS in wild-type mice may involve down-regulation of IL-10 . This anti-inflammatory cytokine, known for its hepatoprotective effects, is secreted simultaneously with proinflammatory cytokines . IL-10 may also limit alcohol-induced liver damage by counteracting the effects of proinflammatory cytokines.

Hong Kong Med J, 1999 Jun, 5(2), 169 - 174
Extragastroduodenal conditions associated with Helicobacter pylori infection; Tsang KW et al.; Helicobacter pylori is a Gram-negative bacterium that is considered a causative agent of peptic ulcer disease, gastric lymphoma, and gastric carcinoma . Helicobacter pylori triggers an intense leucocyte infiltration of the gastric submucosa, an action which is mediated by pro-inflammatory cytokines . This pathogenetic mechanism is common to many other diseases and consequently, Helicobacter pylori seroprevalence has also been investigated in other diseases . It is now known that Helicobacter pylori seropositivity is associated with various cardiovascular, respiratory, extragastroduodenal digestive, neurological, dermatological, autoimmune, and growth disorders . Although the precise role of Helicobacter pylori is unknown in these diseases, the organism can be eradicated using simple and reliable drug regimens . The conditions associated with Helicobacter pylori seropositivity are highlighted in this article.

Ned Tijdschr Geneeskd, 2002 Jan 12, 146(2), 73 - 6
{Meningitis after a superficial dog bite}; Kampinga GA et al.; A 63-year-old healthy man developed acute meningitis . A Gram-stain of the cerebrospinal fluid showed Gram-negative rods, which grew slowly . They were identified by 16S ribosomal RNA sequence-analysis as Capnocytophaga canimorsus, an oral commensal found in various animal species including dogs . Upon further questioning, the patient mentioned a superficial dog bite . Using fluorescence-in situ-hybridisation with specific DNA probes, C . canimorsus cells were detected in a gingiva swab from his dog . The strains isolated from the patient and his dog were identical . The patient made a quick recovery following therapy with cefotaxime . Infections with C . canimorsus are associated with immune suppression (especially splenectomy or alcohol abuse), yet 40% of the patients have no predisposing conditions . Documented infections concern mainly sepsis or meningitis, with a mortality of approximately 30% . Due to its fastidious growth, C . canimorsus may be missed in standard culture methods . Therefore, in each case of unexplained sepsis or meningitis contact with animals should be enquired about.

J Antimicrob Chemother, 2002 Feb, 49(2), 395 - 8
Meropenem stability to beta-lactamase hydrolysis and comparative in vitro activity against several beta-lactamase-producing Gram-negative strains; Franceschini N et al.; The interaction between meropenem and class A, B, C and D beta-lactamases was studied by a spectrophotometric method . Class A, C and D beta-lactamases were unable to confer in vitro resistance to carbapenems . Surprisingly, several class B metallo-beta-lactamases expressed in Escherichia coli failed to confer resistance when a conventional inoculum (105 cfu/mL) was used.

Bioorg Med Chem Lett, 2002 Feb 11, 12(3), 357 - 60
Sequestration of bacterial lipopolysaccharide by bis(Args) gemini compounds; David S et al.; Gemini compounds of the type N(alpha),N(omega)-bis(N(alpha)-lauroyl arginine)alpha,omega-alkylenediamides or bis(Args) bind bacterial lipopolysaccharide and neutralize endotoxic activity in in vitro tumor necrosis factor-alpha and nitric oxide release assays . Sequestration of lipopolysaccharide results in protection in a murine model of endotoxemia . However, the bis(Args) compounds are cytotoxic by virtue of being highly membrane-active . The development of less surface-active analogues may yield potentially therapeutically useful compounds for the treatment of Gram-negative sepsis.

Am J Ind Med, 2002 Feb, 41(2), 111 - 8
Follow-up study of respiratory health of newly-hired female cotton textile workers; Wang XR et al.; BACKGROUND: Numerous studies have investigated adverse effects of exposure to cotton dust on respiratory health, but very limited longitudinal data are available with regard to the early pulmonary response to cotton dust . Moreover, the adverse effects of occupational exposure to cotton dust have been difficult to separate from the confounding effects of smoking . This setting provided a unique opportunity to evaluate early respiratory effects in newly hired and non-smoking female textile workers . METHODS: To identify early pulmonary responses to cotton dust exposure and associated gram-negative bacterial endotoxin, respiratory symptoms and pulmonary function in 225 newly-hired textile workers were assessed at work initiation, and at three and twelve months later . RESULTS: All the workers were females and nonsmokers, with an average age of 18 years . Symptom incidence at three months was 3.6% for usual cough with phlegm, and 6.7% for usual dry cough . Lung function changes were detectable at one year: FEV1 declined by 70 ml and FVC by 124 ml over the year, and workers reporting respiratory symptoms at three months showed a significantly greater cross-shift drop in FEV1 (- 2.3%) than those without the symptoms (- 0.7%) . CONCLUSIONS: These results suggest that the occurrence of respiratory symptoms represents the earliest response to cotton dust exposure, followed by lung function changes . Early respiratory symptoms may be a risk factor for subsequent loss of pulmonary function in cotton textile workers .

J Infect Chemother, 2001 Dec, 7(4), 228 - 38
Analysis of intractable factors in chronic airway infections: role of the autoimmunity induced by BPI-ANCA; Ohtami S et al.; The role of anti-neutrophil cytoplasmic autoantibodies against bactericidal/permeability-increasing protein (BPI-ANCA) in chronic airway infections was investigated . The serum BPI-ANCA titer was correlated with the severity of clinical symptoms in patients with chronic airway infections (P < 0.01), and the serum BPI-ANCA titer decreased with the improvement of the clinical picture, compared with its deterioration (P < 0.05) . The serum BPI-ANCA titer was significantly higher in patients with far-advanced lesions on chest X-rays than in patients with milder lesions (P < 0.01) and in patients with reduced respiratory function (P < 0.05) . Also, the serum BPI-ANCA titer was significantly higher in patients with prolonged colonization of gram-negative bacteria than in those without prolonged gram-negative bacterial colonization (P < 0.05) . When neutrophils from healthy volunteers were incubated with BPI-ANCA before stimulation with lipopolysaccharide (LPS), neutrophil elastase levels decreased in a dose-dependent manner (P < 0.01) . The phagocytic activity of neutrophils was significantly inhibited by BPI-ANCA in a dose-dependent manner (P < 0.01) . The above findings suggest that BPI-ANCA, an autoimmune factor, appears during the course of chronic airway infections, and that this autoimmune factor may make chronic airway infections more intractable, by inhibiting the phagocytic activity of neutrophils for gram-negative bacteria.

J Cell Physiol, 2002 Jan, 190(1), 101 - 8
Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-alpha, IL-1, and RANKL; Suda K et al.; Lipopolysaccharide (LPS), a cell component of Gram-negative bacteria, is a pathogen of inflammatory bone loss . To examine the effects of LPS on the survival and fusion of osteoclasts, mononuclear osteoclasts (preosteoclasts, pOCs) were collected from a mouse co-culture system and cultured in the presence or absence of LPS . Most pOCs died within 24 h in the absence of any stimulus . LPS as well as receptor activator of NF-kappaB ligand (RANKL) supported the survival of pOCs, and induced their fusion to form multinucleated cells (MNCs) . Like authentic osteoclasts, MNCs induced by LPS expressed calcitonin receptors, and formed actin rings on culture plates . LPS-induced MNC formation in pOC cultures was observed even in the presence of osteoprotegerin and interleukin (IL)-1-receptor antagonists . MNC formation was also stimulated by LPS in pOC cultures prepared from tumor necrosis factor (TNF)-receptor-I or TNF-receptor-II deficient mice . LPS induced the degradation of IkappaB in pOCs within 20 min . Lactacystin, an inhibitor of NF-kappaB activation, and wortmannin, an inhibitor of phosphatidylinositol-3 kinase, strongly inhibited LPS-induced MNC formation in pOC cultures . LPS induced pit-forming activity of pOCs in the presence of macrophage-colony stimulating factor (M-CSF) . These findings suggest that LPS stimulates the survival and fusion of pOCs, independent of RANKL, IL-1 or TNF-alpha action . Activation of NF-kappaB and phosphatidylinositol-3 kinase appeared to be involved in LPS-induced effects on pOCs . These observations suggest that LPS is involved directly in inflammatory bone loss, and also indirectly through the production of LPS-induced host factors such as IL-1 and TNF-alpha .

J Endod, 2002 Jan, 28(1), 5 - 7
Leakage evaluation of root end filling materials using endotoxin; Tang HM et al.; Mineral trioxide aggregate (MTA) has been shown to possess excellent sealing ability when tested with dye, bacteria, and a fluid filtration technique . Endotoxin, a component of the cell wall of Gram-negative bacteria, has been proposed to play a role in the pathogenesis of periradicular lesions . This study used a modified Limulus Amebocyte Lysate test for the presence of endotoxin as a tracer and compared the sealing ability of Super-EBA, IRM, amalgam, and MTA . The results showed that MTA permitted less endotoxin leakage than IRM and amalgam at 1, 2, 6, and 12 wk (p < 0.05), and leaked less than Super-EBA at 2 and 12 wk (p < 0.05).

Curr Opin Crit Care, 2001 Dec, 7(6), 401 - 8
Aminoglycoside nephrotoxicity: do time and frequency of administration matter?
Beauchamp D, Labrecque G.
Aminoglycosides remains the mainstay in the treatment of gram-negative infections despite their potential oto-and nephrotoxicity although alternatives with equal or better efficacy are available . Several approaches were investigated to decrease aminoglycosides nephrotoxicity . Among them, only the once-daily dosing of aminoglycosides has been brought to the clinic and physicians are now increasingly adopting this approach to reduce the toxicity of these agents . The incidence of aminoglycoside nephrotoxicity can be further reduced in view of the recent data on the circadian variations of their nephrotoxicity . In fact, it has been clearly demonstrated in both experimental animals and humans that the toxicity is maximal when the drug is injected during the rest period compared with the activity period . Thus, injecting aminoglycosides once-daily at the time of the lowest toxicity is actually the most interesting and clinically applicable approach to reduce aminoglycosides toxicity.

Bone Marrow Transplant, 2001 Dec, 28(12), 1129 - 34
Infections during mobilizing chemotherapy and following autologous stem cell transplantation; Toor AA et al.; Autologous peripheral blood stem cells (PBSC), for transplantation following high-dose chemotherapy, are collected using regimens containing cytokines with or without chemotherapy . The added period of neutropenia prior to stem cell transplantation (SCT) in patients receiving chemotherapy mobilization may increase the risk of infections following transplantation . We studied the incidence of culture-positive infections in 107 consecutive patients who were divided into three groups, according to whether they experienced extended neutropenia during chemotherapy for stem cell mobilization as well as post autotransplant . All the patients received antibiotic prophylaxis and hematopoietic growth factors during neutropenia . The total duration of pre-transplant neutropenia differed among the three mobilization schemes (growth factors alone; one cycle; or two cycles of chemotherapy plus growth factor for mobilization) at 0, 6 and 18 days, respectively (median) . However the post-autograft time to myeloid engraftment was similar at 10 days (median) . The incidence of culture-proven infections in all three groups was similar . Using fluconazole for yeast prophylaxis, 40% patients developed gastrointestinal colonization with yeast, and the majority of speciated isolates were Candida glabrata . Bacteremia developed in 22% and 9% of patients with S . epidermidis and Gram-negative organisms, respectively, while 11% developed C . difficile-associated diarrhea . In conclusion, treatment using none, one or two cycles of mobilizing chemotherapy pre-transplant does not influence the overall incidence of infections among autologous SCT recipients . However, although post-transplant neutropenia is brief, infections remain a significant cause of morbidity.

Blood Purif, 2002, 20(1), 55 - 60
Bioartificial kidney alters cytokine response and hemodynamics in endotoxin-challenged uremic animals; Fissell WH et al.; The mortality from sepsis complicated by renal failure remains extremely high despite the application of modern renal replacement therapy . This study investigated whether treatment with a bioartificial kidney consisting of a hemofilter in a continuous venovenous hemofiltration circuit (CVVH) with a cartridge containing renal proximal tubule cells, also called the Renal Tubule Assist Device (RAD), would alter the course of sepsis in an animal model . The RAD has been previously characterized in vitro and ex vivo and provides transport, metabolic and endocrine activity . Mongrel dogs (n = 10) underwent surgical nephrectomy and 48 h later were treated with CVVH and either a RAD containing cells (n = 5) or an identically prepared sham cartridge (n = 5) . After 4 h of therapy, intravenous endotoxin 2 mg/kg was infused over 1 h to simulate gram-negative septic shock . Data on blood pressure, cardiac output and systemic markers of inflammation were collected . Mean peak levels of an anti- inflammatory cytokine, IL-10, were significantly higher in cell-treated animals (15.25 vs . 6.29 ng/ml; p = 0.037), and mean arterial pressures were higher in cell-treated versus sham-treated animals (p < 0.04) . We have demonstrated that treatment of an animal model of endotoxin shock and renal failure with a bioartificial kidney has measurable effects on circulating mediators of inflammation and on hemodynamic stability of the challenged animal .

Biochemistry, 2002 Jan 29, 41(4), 1174 - 81
Catalytic mechanism of CMP:2-keto-3-deoxy-manno-octonic acid synthetase as derived from complexes with reaction educt and product; Jelakovic S et al.; The activation of the sugar 2-keto-3-deoxy-manno-octonic acid (Kdo) is catalyzed by CMP-Kdo synthetase (EC 2.7.7.38) and results in a monophosphate diester with CMP . The enzyme is a pharmaceutical target because CMP-Kdo is required for the biosynthesis of lipopolysaccharides that are vital for Gram-negative bacteria . We have established the structures of an enzyme complex with the educt CTP and of a complex with the product CMP-Kdo by X-ray diffraction analyses at 100 K, both at 2.6 A resolution . The N-terminal domains of the dimeric enzyme bind CTP in a peculiar nucleotide-binding fold with the beta- and gamma-phosphates located at the so-called "PP-loop", whereas the C-terminal domains participate in Kdo binding and in the dimer interface . The unstable nucleotide-sugar CMP-Kdo was produced in a crystal and stabilized by freezing to 100 K . Its formation is accompanied by an induced fit involving mainchain displacements in the 2 A range . The observed binding conformations together with the amino acid conservation pattern during evolution and the putative location of the required Mg(2+) ion suggest a reaction pathway . The enzyme is structurally homologous to the CMP-N-acetylneuraminic acid synthetases in all parts except for the dimer interface . Moreover, the chainfold and the substrate-binding positions resemble those of other enzymes processing nucleotide sugars.

J Immunol, 2002 Feb 1, 168(3), 1286 - 93
In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation; Wolfs TG et al.; The reported requirement of functional Toll-like receptor (TLR)4 for resistance to Gram-negative pyelonephritis prompted us to localize the expression of TLR2 and TLR4 mRNA in the kidney at the cellular level by in situ hybridization . The majority of the constitutive TLR2 and TLR4 mRNA expression was found to be strategically located in the renal epithelial cells . Assuming that the TLR mRNA expression is representative of apical protein expression, this suggests that these cells are able to detect and react with bacteria present in the lumen of the tubules . To gain insight in the regulation of TLR expression during inflammation, we used a model for renal inflammation . Renal inflammation evoked by ischemia markedly enhanced synthesis of TLR2 and TLR4 mRNA in the distal tubular epithelium, the thin limb of Henle's loop, and collecting ducts . The increased renal TLR4 mRNA expression was associated with significant elevation of renal TLR4 protein expression as evaluated by Western blotting . Using RT-PCR, the enhanced TLR2 and TLR4 mRNA expression was shown to be completely dependent on the action of IFN-gamma and TNF-alpha . These results indicate a potential mechanism of increased immunosurveillance during inflammation at the site in which ascending bacteria enter the kidney tissue, i.e., the collecting ducts and the distal part of the nephron.

Pharmacotherapy, 2002 Jan, 22(1), 105 - 8
Sudden irreversible sensory-neural hearing loss in a patient with diabetes receiving amikacin as an antibiotic-heparin lock; Saxena AK et al.; Gram-negative septicemia due to central venous catheter-related infection is a leading cause of mortality and morbidity among patients who undergo hemodialysis . Antibiotic-heparin locks are valuable for preserving access sites and lowering the cost and inconvenience associated with central venous catheter replacement and surgical interventions . The optimal duration of use of an antibiotic-heparin lock is unknown . Prolonged use of an amikacin-heparin lock may lead to severe irreversible sensory-neural hearing loss . Patients at risk for this complication should be monitored for its emergence to facilitate early detection . A 43-year-old man with diabetic end-stage renal disease received hemodialysis through a permanent catheter . After 16 weeks of using an amikacin-heparin lock, he suddenly developed sensory-neural hearing loss of 40 dB, which affected high frequencies . His condition progressed relentlessly within 1 week despite immediate discontinuation of the amikacin-heparin lock . The patient developed severe irreversible hearing loss below 80 dB for both high and low frequencies.

J Trauma, 2002 Jan, 52(1), 13 - 7
The effects of hemodynamic shock and increased intra-abdominal pressure on bacterial translocation; Doty JM et al.; BACKGROUND: We hypothesized that hemorrhagic shock followed by the abdominal compartment syndrome (ACS) resulted in bacterial translocation (BT) from the gastrointestinal (GI) tract . METHODS: Nineteen Yorkshire swine (20-30 kg) were divided into two groups . In the experimental group, group 1 (n = 10), animals were hemorrhaged to a mean arterial pressure (MAP) of 25-30 mm Hg for a period of 30 minutes and resuscitated to baseline MAP . Subsequently, intra-abdominal pressure (IAP) was increased to 30 mm Hg above baseline by instilling sterile normal saline into the peritoneal cavity . The IAP was maintained at this level for 60 minutes . Acid/base status, gastric mucosal ph (pHi), superior mesenteric artery (SMA) blood flow, and hemodynamic parameters were measured and recorded . Blood samples were analyzed by polymerase chain reaction (PCR) for the presence of bacteria . Spleen, lymph node, and portal venous blood cultures were obtained at 24 hours . Results were analyzed by ANOVA and are reported as mean +/- SEM . The second group was the control . These animals did not have the hemorrhage, resuscitation, or intra-abdominal hypertension (IAH) but were otherwise similar to the experimental group in terms of laparotomy and measured parameters . RESULTS: SMA blood flow in group 1 (baseline of 0.87 +/- 0.10 l/min) decreased in response to hemorrhage (0.53 +/- 0.10 l/min, p = 0.0001) and remained decreased with IAH (0.63 l/min +/- 0.10, p = 0.0006) as compared to control and returned towards baseline (1.01 +/- 0.5 l/min) on relief of IAH . pHi (baseline of 7.21 +/- 0.03) was significantly decreased with hemorrhage (7.04 +/- 0.03, p = 0.0003) and decreased further after IAH (6.99 +/- 0.03, p = 0.0001) in group 1 compared to control, but returned toward baseline at 24 hours (7.28 +/- 0.04) . The mean arterial pH decreased significantly from 7.43 +/- 0.01 at baseline to 7.27 +/- 0.01 at its nadir within group 1 (p = 0.0001) as well as when compared to control (p = 0.0001) . Base excess was also significantly decreased between groups 1 and 2 during hemorrhage (3.30 +/- 0.71 vs . 0.06 +/- 0.60, p = 0.001) and IAH (3.08 +/- 0.71 vs . -1.17 +/- 0.60, p = 0.0001) . In group 1, 8 of the 10 animals had positive lymph node cultures, 2 of the 10 had positive spleen cultures, and 2 of the 10 had positive portal venous blood cultures for gram-negative enteric bacteria . Only 2 of the 10 animals had a positive PCR . In group 2, five of the nine animals had positive lymph node cultures, zero of the nine had positive spleen cultures, and one of the nine had positive portal venous blood cultures . Two of the nine animals had positive PCRs . There was no significant difference in cultures or PCR results between the two groups (Fisher's exact test, p = 0.3) . CONCLUSION: In this study, hemorrhage followed by reperfusion and a subsequent insult of IAH caused significant GI mucosal acidosis, hypoperfusion, as well as systemic acidosis . These changes did not appear to be associated with a significant bacterial translocation as judged by PCR measurements, tissue, or blood cultures.

J Bacteriol, 2002 Feb, 184(3), 754 - 9
Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity; Cascales E et al.; The Tol-Pal system of gram-negative bacteria is composed of five proteins . TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane . In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli . lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles . In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain . Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes . The density of Pal was measured and found to be lower than that of Lpp . However, Pal was present in larger amounts compared to TolA and TolR proteins . The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

J Bacteriol, 2002 Feb, 184(3), 695 - 705
Analysis of ftsQ mutant alleles in Escherichia coli: complementation, septal localization, and recruitment of downstream cell division proteins; Chen JC et al.; FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli . To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature . Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting . The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature . FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum . These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery . Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

Am J Respir Crit Care Med, 2002 Jan 15, 165(2), 171 - 5
Lung tissue concentrations of nebulized amikacin during mechanical ventilation in piglets with healthy lungs; Goldstein I et al.; The tissue concentration of aminoglycosides in lung parenchyma is the main determinant of bactericidal efficiency . The aim of the study was to compare the lung deposition of amikacin administered either by an ultrasonic nebulizer or by intravenous infusion during mechanical ventilation . Eighteen healthy ventilated piglets received a single daily dose of amikacin by intravenous infusion (15 mg . kg(-1)) and 18 by aerosol (1 g in 12 ml) . The amount of aerosolized amikacin reaching the tracheobronchial tree represented 40 +/- 5% of the initial dose with an aerodynamic size distribution showing 50% of particles ranging between 0.5 and 5 microm mass median diameter . Animals were killed at different time intervals after the second dose . Tissue concentrations of amikacin were determined on cryomixed multiple lung specimen by an immunoenzymatic method . The lung concentrations of nebulized amikacin, peaking at 208 +/- 76 microg . g(-1), were more than 10-fold higher than the lung concentrations of intravenous amikacin and were homogeneously distributed throughout the lung parenchyma . Amikacin plasma concentrations lower than 5 mmol . l(-1) were measured after the sixth hour after the nebulization . In conclusion, the ultrasonic nebulization of amikacin resulted in high tissue concentrations, far above the minimal inhibitory concentrations of most gram-negative strains.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 257 - 62
Comparative evaluation of different chromogenic/fluorogenic media for detecting Escherichia coli O157:H7 in food; Manafi M et al.; Escherichia coli O157:H7 is a serious and common human pathogen that can cause diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome (HUS) . This study evaluated the enrichment, detection and confirmation procedures for the isolation of E . coli O157:H7 from raw ground beef and raw drinking milk . The purpose of this investigation was to compare Rainbow Agar O157 (RB; Biolog, Hayward, USA), Biosynth Culture Medium O157:H7 (BCM O157:H7; Biosynth, Staad, Switzerland) and Fluorocult HC (HC; Merck, Darmstadt, Germany) with the conventional Sorbitol MacConkey Agar (SMAC, Merck) using mEC + n (raw ground beef) and mTSB + n (raw milk) enrichment media . Single-path GLISA test (Gold Labeled Immuno Sorbent Assay; Merck) was used as the confirmation test . Growth of 466 strains of gram-negative rods isolated from food samples and 46 known E . coli strains from type culture and other collections (34 E . coli O157:H7 strains and 12 serotypes other than E . coli O157:H7) was examined on the agar media . The E . coli O157:H7 strains could readily be isolated and recognized uniquely by their typical black/grey colonies on RB and blue/black colonies on BCM O157:H7 . Examination of the 46 known strains of E . coli reference strains showed false negative results on BCM O157:H7 (3.0%), RB (8.8%), HC (5.9%) and SMAC (5.9%) agars . On BCM O157:H7 no false negative results were found with the typical E . coli O157:H7 (beta-D-glucuronidase and sorbitol negative strains) . One of two atypical E . coli O157:H7 strains (beta-D-glucuronidase positive) showed similar colouration to the typical strains and was mis-identified by each of the three media (RB, BCM O157:H7, and SMAC agar media) . None of the 60 food samples tested yielded E . coli O157:H7 . Examination of the food samples, showed that RB gave the lowest number of false positives . The percentages were RB (2.1%), BCM O157:H7 (3.3%), HC (6.2%), and SMAC (57.3%).

Pituitary, 2000 Dec, 3(4), 227 - 30
Hepatolithiasis (intrahepatic stone) during octreotide therapy for acromegaly: a case report; Sheehan MT et al.; We report a case of hepatolithiasis (intrahepatic stone) complicated by gram-negative sepsis in a 37 year old male with acromegaly being treated with octreotide . As a child, he had suffered a traumatic injury to his liver requiring the surgical repair of a laceration . This is the first reported case of hepatolithiasis during octreotide therapy . Gallstones and bile sludge are common side effects of octreotide therapy but rarely become symptomatic or require treatment . Hepatolithiasis is uncommon in western countries but is quite prevalent in East Asia and is often associated with a predisposing condition that causes intrahepatic bile stasis (eg . bile duct stricture) . In addition to its known effect on gallbladder stasis, octreotide alters bile acid composition and may thus hasten intrahepatic sludge and stone formation . Extra caution should be taken in using octreotide or its long-acting analog in patients otherwise predisposed to intrahepatic bile stasis.

Infection, 2001 Dec, 29(6), 345 - 7
Spontaneous gram-negative cellulitis in a liver transplant recipient; Paterson DL et al.; A 47-year-old liver transplant recipient developed fever and cellulitis on the 8th post-transplant day . The clinical appearances were of a rapidly advancing cellulitis . The patient had a past history of severe peripheral edema and hypoalbuminemia . Blood cultures and skin biopsy grew Escherichia coli . To our knowledge, this is the first reported case of E . coli cellulitis in a liver transplant recipient . However, cases have previously been described in patients with cirrhosis or idiopathic nephritic syndrome, conditions which share predisposing features of peripheral edema and hypoalbuminemia . Bacteremic gram-negative cellulitis should be considered in compromised patients with unusual presentations of cellulitis.

J Physiol Pharmacol, 2001 Dec, 52(4 Pt 1), 611 - 23
Central and peripheral neural aspects of gastroprotective and ulcer healing effects of lipopolysaccharides; Konturek PC et al.; Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria playing a central role as potent endotoxins in the pathogenesis of endotoxic shock . Although large amounts of endotoxin may produce hemorrhagic lesions in the stomach, the possible gastroprotective effect of central or peripheral LPS against the acute gastric lesions has not been extensively studied . The aim of the present study was to compare the effect of intracerebroventricular (i.c.v.) and parenteral (i.p.) injection of LPS against gastric lesions induced by 100% ethanol . Male Wistar rats were treated either with a) vehicle (control); b) E-coli-LPS in various concentrations (1-10 microg/kg i.c.v or 0.1-40 mg/kg i.p.) followed 30 min later by 100% ethanol . The effects of pretreatment with nonselective inhibitor of nitric oxide synthase (L-NAME, 20 mg/kg i.g.) or selective inhibitor of inducible nitric oxide synthase, L-NIL (30 mg/kg i.g.) on the gastroprotection induced by LPS was investigated . One hour after ethanol application, the gastric blood flow (GBF) and the area of gastric lesions were determined . In addition, the mucosal expression of iNOS, cNOS and leptin was assessed using RT-PCR . LPS applied i.c.v . or i.p . dose dependently reduced gastric lesions induced by ethanol and this effect was similar to that observed after the administration of NO donor (SNAP) . LPS-induced protection was significantly abolished by L-NAME and significantly attenuated by the selective inhibitor of iNOS (L-NIL) . The expression of cNOS was detected in vehicle treated gastric mucosa and did not change after LPS administration . iNOS was not detectable in intact mucosa but its expression dose-dependently increased after the LPS administration . The i.c.v . administration of LPS did not upregulate further the iNOS expression, and dose-dependently inhibited the leptin mRNA expression in gastric mucosa . We conclude that LPS applied centrally or peripherally protects gastric mucosa against ethanol-induced damage through an increase in gastric microcirculation mediated by NO due to overexpression of iNOS . Transcriptional downregulation of leptin in gastric mucosa is probably due to the increased leptin release induced by the intracerebroventricular application lipopolysaccharide.

J Biol Chem, 2002 Mar 15, 277(11), 9077 - 87 Epub 2002 Jan 10.
A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production; Watters JJ et al.; Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia . Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities . Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types . Because the mitogen-activated protein kinases ERK-1 and ERK-2 have been implicated in the control of many immune responses, we tested the concept that they are a key indicator for differences in cellular LPS sensitivity . We observed that murine RAW 264.7 macrophages and murine BV-2 microglial cells both respond to LPS by exhibiting increased IkappaBalpha degradation, enhanced NF-kappaB DNA binding activity, and elevated nitric oxide and interleukin-1beta production . Although LPS potently stimulates ERK activation in RAW 264.7 macrophages, it does not activate ERK-1/-2 in BV-2 microglia . Moreover, antagonism of the MEK/ERK pathway potentiates LPS-stimulated nitric oxide production, suggesting that LPS-stimulated ERK activation can exert inhibitory effects in macrophage-like cells . These data support the idea that ERK activation is not a required function of LPS-mediated signaling events and illustrate that alternative/additional pathways for LPS action exist in these cell types.

Bioelectrochemistry, 2002 Jan, 55(1-2), 161 - 4
Effects of low-frequency magnetic fields on bacteria Escherichia coli; Strasak L et al.; The effects of low-frequency magnetic fields (Bm=2.7-10 mT, f=50 Hz, time of exposure t=0-12 min, laboratory temperature) on the viability and oxidoreductive activity of gram-negative bacteria Escherichia coli were investigated . The growth of these bacteria was negatively affected by such fields . We compared two experimental systems--solenoid {Sb . Lek . 99 (1998) 455} and a cylindrical spool--to find differences between nonhomogeneous and "more homogeneous" magnetic fields . We observed analogous effects in both experimental conditions . The growth curve of the exposed bacteria was lower than the control one . The ability of bacteria to form colonies decreased with increasing magnetic field intensity and with increasing time of exposure . The oxidoreductive activity was measured using reduction of a tetrazolium salt . The decrease in oxidoreductive activity with increasing time of exposure was observed, but the effect was due to a lower amount of bacteria surviving the exposure to the magnetic fields . The decrease in oxidoreductive activity and ability to form colonies were compared with the assumption that the effect of magnetic field is probably bactericidal.

Mikrobiol Z, 2001 Sep-Oct, 63(5), 10 - 20
{Isolation and characterization of Cytophaga lytica lipopolysaccharide}; Varbanets LD et al.; Lipopolysaccharide (LPS) and its structural components: lipid A, O-specific polysaccharide (O-PS) and oligosaccharide core (OG-core) have been isolated from Cytophaga lytica . 3-Oxytetradecanoic (40.8%) and dodecanoic (28.7%) are the predominant fatty acids of lipid A; pentadecanoic (6.8%), 3-oxyhexadecanoic (6.5%) as well as hexadecanoic (5.4%) acids have been found as well . The content of the rest of fatty acids is inconsiderable (2.3 to 0.5%) . OG-core contained monosaccharides both typical of the most Gram-negative bacteria (glucose, mannose, galactose, rhamnose, glucosamine) and rarely occurring one-arabinose . O-PS is represented by glucose, mannose, rhamnose, glucosamine, galactosamine as well as unidentified hexosamine.

Crit Care Med, 2002 Jan, 30(1 Suppl), S64 - 73
Endotoxin tolerance: a review; West MA et al.; Endotoxin tolerance was initially described when it was observed that animals survived a lethal dose of bacterial endotoxin if they had been previously treated with a sublethal injection . In animal models, two phases of endotoxin tolerance are described, an early phase associated with altered cellular activation and a late phase associated with the development of specific antibodies against the polysaccharide side chain of Gram-negative organisms . Recently, there has been a tremendous resurgence of interest in the mechanisms responsible for altered responsiveness to bacterial endotoxin . Host immune cells, particularly macrophages and monocytes, that are exposed to endotoxin for 3 to 24 hrs are rendered "tolerant" and manifest a profoundly altered response when rechallenged with bacterial endotoxin or lipopolysaccharide . The "lipopolysaccharide-tolerant" phenotype is characterized by inhibition of lipopolysaccharide-stimulated tumor necrosis factor production, altered interleukin-1 and interleukin-6 release, enhanced cyclooxygenase-2 activation, inhibition of mitogen-activated protein kinase activation, and impaired nuclear factor-kappa B translocation . Human monocytes and macrophages can be induced to become tolerant, and there is increasing evidence that monocytic cells from patients with systemic inflammatory response syndrome and sepsis have many characteristics of endotoxin tolerance.

Nature, 2001 Dec 20-27, 414(6866), 920 - 4
MIF regulates innate immune responses through modulation of Toll-like receptor 4; Roger T et al.; Macrophages are pivotal effector cells of the innate immune system, which is vital for recognizing and eliminating invasive microbial pathogens . When microbial products bind to pathogen-recognition receptors, macrophages become activated and release a broad array of cytokines that orchestrate the host innate and adaptive immune responses . Initially identified as a T-cell cytokine, macrophage migration inhibitory factor (MIF) is also a macrophage cytokine and an important mediator of inflammation and sepsis . Here we report that MIF is an essential regulator of macrophage responses to endotoxin (lipopolysaccharide) and Gram-negative bacteria . Compared with wild-type cells, MIF-deficient macrophages are hyporesponsive to lipopolysaccharide and Gram-negative bacteria, as shown by a profound reduction in the activity of NF-kappaB and the production of tumour-necrosis factor-alpha . This reduction is due to a downregulation of Toll-like receptor 4 (TLR4), the signal-transducing molecule of the lipopolysaccharide receptor complex, and is associated with decreased activity of transcription factor PU.1, which is required for optimal expression of the Tlr4 gene in myeloid cells . These findings identify an important role for MIF in innate immunity and provide a molecular basis for the resistance of MIF-deficient mice to endotoxic shock.

Ther Apher, 2001 Oct, 5(5), 326 - 34
A new endotoxin adsorber: first clinical application; Ullrich H et al.; In an open, uncontrolled pilot study, 5 men and 1 woman with suspected gram-negative sepsis were treated with a new whole-blood endotoxin adsorption system . Lipopolysaccharide (LPS) adsorption was carried out by hemoperfusion over high-affinity polymethacrylate-bound albumin (Fresenius Endotoxin Adsorber EN 500) . All patients suffered from endotoxemia (>20 pg/ml LAL) and met at least two systemic inflammatory response syndrome (SIRS) criteria . Four patients suffered from pneumonia due to mechanical ventilation, one from peritonitis, and one from pneumonia and peritonitis . Endotoxin adsorption was very well tolerated, and efficient LPS removal was shown in all patients . Apache II score immediately before immunoadsorption was 23.5 and was 22.3 after the last treatment . All 6 critically ill patients improved substantially and were discharged from the intensive care unit . LPS whole blood immunoadsorption is a promising new method . No side effects have been observed thus far . A large controlled study to prove clinical efficacy in patients with severe sepsis is under way.

Transfusion, 2001 Dec, 41(12), 1493 - 9
Transfusion-transmitted bacterial infection in the United States, 1998 through 2000; Kuehnert MJ et al.; BACKGROUND: Bacterial contamination of blood components can result in transfusion-transmitted infection, but the risk is not established . STUDY DESIGN AND METHODS: Suspected cases of transfusion-transmitted bacteremia were reported to the CDC by participating blood collection facilities and transfusion services affiliated with the American Red Cross, AABB, or Department of Defense blood programs from 1998 through 2000 . A case was defined as any transfusion reaction meeting clinical criteria in which the same organism species was cultured from a blood component and from recipient blood, with the organism pair confirmed as identical by molecular typing . RESULTS: There were 34 cases and 9 deaths . The rate of transfusion-transmitted bacteremia (in events/million units) was 9.98 for single-donor platelets, 10.64 for pooled platelets, and 0.21 for RBC units; for fatal reactions, the rates were 1.94, 2.22, and 0.13, respectively . Patients at greatest risk for death received components containing gram-negative organisms (OR, 7.5; 95% CI, 1.3-64.2; p = 0.009) . CONCLUSION: Bacterial contamination of blood is an important cause of transfusion-transmitted infection; infection risk from platelet transfusion is higher compared with that from RBCs, and, overall, the risk of infection from bacterial contamination now may exceed that from viral agents . Recipients of components containing gram-negative organisms are at highest risk for transfusion-related death . The results of this study may help direct efforts to improve transfusion-related patient safety.

J Microbiol Methods, 2002 Feb, 48(2-3), 139 - 47
Flash detection/identification of pathogens, bacterial spores and bioterrorism agent biomarkers from clinical and environmental matrices; White DC et al.; We propose to develop an integrated rapid, semiportable, prototype point microbial detection/identification system for clinical specimens that is also capable of differentiating microbial bioterrorism attacks from threats or hoaxes by defining the pathogen . The system utilizes "flash" extraction/analytical system capable of detection/identification of microbes from environmental and clinical matrices . The system couples demonstrated technologies to provide quantitative analysis of lipid biomarkers of microbes including spores in a system with near-single cell (amol/microl) sensitivity . Tandem mass spectrometry increases specificity by providing the molecular structure of neutral lipids, phospholipids, and derivatized spore-specific bacterial biomarker, 2,6-dipicolinic acid (DPA) as well as the lipopolysaccharide-amide-linked hydroxy-fatty acids (LPS-ALHFA) of Gram-negative bacteria . The extraction should take about an hour for each sample but multiple samples can be processed simultaneously.

Expert Opin Investig Drugs, 2001 Jul, 10(7), 1231 - 41
Helicobacter pylori: strategies for treatment; Gomollon F et al.; Helicobacter pylori (Hp) is a Gram-negative bacteria able to live in the human stomach, a very surprising fact considering the acid environment of gastric mucosa . Identified by Marshall and Warren in 1982 {1,2}, this bacterium seems aetiologically related to many gastric diseases, previously known as 'acid related diseases' . Compelling evidence demonstrates that Hp is the most important aetiological agent of gastritis {3}, the principal causal factor in peptic ulcer {4}, contributes to the genesis of gastric cancer {5} and has a critical role in the development of many mucosa-associated lymphoid tissue (MALT) lymphomas {6} . Although experimental data have recently provided hard evidence to support the role of Hp in the genesis of gastritis, ulcer and carcinoma {7}, a critical argument for Hp generating peptic ulcer disease has been, in fact, the change in the natural history of peptic ulcer that follows the cure of the infection.

Mol Biol (Mosk), 2001 Nov-Dec, 35(6), 1105 - 9
{Revealing key interactions in the metabolic network: a model of primary phosphate transport in bacteria}; Titov II et al.; One of the main problems of metabolic engineering is to determine the genetically controlled limiting links of a metabolic network . We have built a model of the primary transport of inorganic phosphates (Pi), analyzed the Pi metabolic network in Gram-negative bacteria, and determined the factors controlling the phosphate exchange . The model explains why the Pi primary transport is not observed at the release stage . The nonlinearity of primary transport and the differences in its parameters in the membrane and within the cell give rise to transport asymmetry, i.e., the Pi release rate is low as compared with the uptake rate, and is small at the background of secondary transport . Discussed is a general scheme of coordination between primary and secondary transport, which are interconnected through the substrate-product reration.

Shock, 2001 Dec, 16(6), 454 - 8
Upregulation of intraepithelial lymphocyte (IEL) function in the small intestinal mucosa in sepsis; Nussler NC et al.; Host defense mechanisms preventing bacterial invasion are particularly important in the gastrointestinal tract, since most gram-negative infections originate from there . Intraepithelial lymphocytes (IEL) seem to play an important role in this immune surveillance of the intestine, although their function in sepsis is not fully understood . To evaluate the characteristics of IEL in sepsis, C57BL/6 mice received a non-lethal dose of LPS and IEL were harvested at various time points thereafter . Although IEL displayed no phenotypic changes after endotoxemia, they displayed enhanced cytolytic activity and increased proliferation after LPS injection In addition, IEL from septic mice showed enhanced gamma interferon (IFN-gamma) production after LPS administration . The production of IFN-gamma may have induced the increased intestinal NOS-2 mRNA expression which was observed after endotoxemia . In conclusion, endotoxemia leads to functional activation of IEL without phenotypic changes . The activation of IEL and the subsequently increased NOS-2 expression may be important mechanisms in maintaining the mucosal barrier after sublethal LPS challenge.

J Bioenerg Biomembr, 2000 Apr, 32(2), 143 - 52
Heme centers of Rhodothermus marinus respiratory chain . Characterization of its cbb3 oxidase; Pereira MM et al.; Rhodothermus (R.) marinus, a thermohalophilic gram-negative, and strict aerobic bacterium, has a rather distinct respiratory chain, containing a caa3 terminal oxidase, a novel cytochrome bc complex and a HiPIP, which is an electron carrier between this complex and a terminal oxidase (Pereira et al (1999a, c) . To further elucidate this unusual respiratory system, its membrane-bound heme centers were characterized by visible and EPR spectroscopies as well as by redox potentiometry . Rhodothermus marinus contains mostly B- and C-type hemes; a small amount of A-type heme is also detected . The heme centers have relatively low reduction potentials, ranging from ca . +250 to -60 mV, at pH 7 . A Rieske-type center was not detected, suggesting the absence of a canonical complex III . The major terminal oxidase expressed by R . marinus is a cbb3-type oxidase . Its presence is in agreement with molecular biology studies, which reveal the existence of a gene encoding for a FixN-type oxidase . The oxidase was partially purified and appears to have five subunits, with apparent molecular masses of 64, 57, 36, 26 (C-type heme subunit), and 13 kDa . It contains two low-spin heme C centers, one high-, and one low-spin heme B centers . A full description of the equilibrium redox behavior of the heme centers was obtained for a cbb3 oxidase for the first time . The optical spectrum for each heme center and the corresponding reduction potentials were determined at pH 7: + 195 (heme C), +120 (heme B), -50 (heme C), and -50 mV (heme B3).

Mol Plant Microbe Interact, 2001 Dec, 14(12), 1351 - 63
Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria; Heeb S et al.; The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads . The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress . A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes . The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain . The periplasmic loop domain of GacS is poorly conserved in diverse bacteria . Thus, a common signal interacting with this domain would be unexpected . Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases . Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level . It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

Dig Dis Sci, 2001 Dec, 46(12), 2768 - 72
Antioxidants inhibit cytokine production and suppress NF-kappaB activation in CAPAN-1 and CAPAN-2 cell lines; Blanchard JA 2nd et al.; Interleukin (IL) -6 and IL-8 are cytokines that have been shown to play a role in several pancreatic diseases, including acute pancreatitis, chronic pancreatitis, and pancreatic adenocarcinoma . Previously, we have demonstrated that tumor necrosis factor-alpha (TNF-alpha) and gram-negative bacterial lipopolysaccharide stimulate production of IL-6 and IL-8 and activation of the transcription factor NF-kappaB in the well-differentiated pancreatic ductal adenocarcinoma cell lines CAPAN-1 and CAPAN-2 . In these studies we have examined the effect of chain-breaking and glutathione-enhancing antioxidants on NF-kappaB activation and production of IL-6 and IL-8 in these cell lines . Generally, suppression of NF-kappaB activation correlated well with inhibition of IL-6 and IL-8 secretion . In the CAPAN-2 cell line, antioxidants inhibited both NF-kappaB activation and IL-6 and IL-8 secretion . In the CAPAN-1 cell line, antioxidants generally failed to suppress both NF-kappaB activation and IL-6 and IL-8 secretion . The single exception was the chain-breaking antioxidant butylated hydroxyanisole (BHA), which markedly inhibited IL-6 and IL-8 secretion, but had no effect on NF-kappaB activation . These findings may have implications for the treatment of acute and chronic pancreatitis and pancreatic cancer.

Eur J Oral Sci, 2001 Dec, 109(6), 402 - 8
The heat shock response of Fusobacterium nucleatum; Skar CK et al.; The heat-shock response of the oral Gram-negative bacterium Fusobacterium nucleatum was examined . Different strains of F . nucleatum were grown at 37 C . 42 degrees C and 48 C in the presence of {35S}methionine . Cellular proteins synthesised after shifts to higher temperatures were analysed by SDS-PAGE and autoradiography . Strains ATCC 10953, F1, F3 and Fev1 exhibited heat-shock response, and major proteins were observed at 60, 70 and 90 kDa . but increased protein synthesis was also observed for other proteins . Immunoblot analysis, using a panel of antibodies directed to epitopes on different known heat-shock proteins revealed cross-reactive proteins, indicating homology between Escherichia coli, Mycobacterium leprae and F . nucleatum heat shock proteins.

J Mol Microbiol Biotechnol, 2002 Jan, 4(1), 11 - 20
Ushers and secretins: channels for the secretion of folded proteins across the bacterial outer membrane; Thanassi DG; Gram-negative bacteria have evolved a number of pathways for extracellular protein secretion . Proteins targeted for secretion in Gram-negative bacteria must cross the outer membrane in addition to the cytoplasmic membrane . This must be accomplished without compromising the barrier properties of the outer membrane and is further complicated by the fact that proteins may fold prior to secretion outside the cell . This review will discuss the usher and secretin families of integral outer membrane proteins, which function to allow the secretion of folded proteins in Gram-negative bacteria.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 19 - 34
Modular organization of the AIDA autotransporter translocator: the N-terminal beta1-domain is surface-exposed and stabilizes the transmembrane beta2-domain; Konieczny MPJ et al.; The adhesin involved in diffuse adherence (AIDA-I) of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27) is synthesized as a precursor molecule . This pre-pro-protein is N- and C-terminally processed to generate three distinct domains, which are characteristic for autotransporter secretion systems in Gram-negative bacteria: the N-terminal pre-peptide, the alpha-domain and the C-terminal beta-domain . The outer membrane-integrated beta-domain (AIDAC) is responsible for the surface-presentation of the alpha-domain (AIDA-I) and is thus termed 'translocator' . Characterization of extracted N-terminally truncated forms and of in vitro refolded proteins revealed a core structure at the C-terminus of the translocator which was found to be very stable even in the presence of SDS . Denaturation occurs only after additional incubation at temperatures above 80 degrees C . Reporter-epitope insertions were used to analyze the location of regions of membrane-integrated AIDAC relative to the membrane . The modified topological model developed for the AIDA translocator suggests the N-terminal domain (beta1) encompasses approximately 10 kDa to represent a completely surface-exposed segment while the C-terminal compact core domain (beta2) remains integrated in the membrane as a beta-barrel-like structure . Though the beta2-core structure alone harbours all the information for the outer membrane integration of AIDAC it is additionally stabilized by the beta1-domain . Access to large amounts of complete as well as truncated AIDAC proteins facilitated the study of protein folding by CD and fluorescence spectroscopy . A potential pore forming activity of the translocator using the completely refolded AIDAC or the beta2-core in black-lipid membranes could not be demonstrated.

Scand J Gastroenterol, 2001 Dec, 36(12), 1239 - 47
Influence of bacterial lipopolysaccharide on healing of chronic experimental ulcer in rat; Konturek PC et al.; BACKGROUND: Lipopolysaccharides (LPS) are major components of the outer membrane of Gram-negative bacteria, which play a central role as potent endotoxins in the pathogenesis of the Gram-negative septicaemia . Although it is well known that large amounts of endotoxin may produce haemorrhagic lesions in the stomach, the effect of LPS on ulcer healing has not been fully clarified . The aim of the present study was to examine the effect of parenteral injection of LPS at different doses on the course of ulcer healing in rats . METHODS: Gastric ulcers were induced in Wistar rats by serosal application of an acetic acid area . After ulcer induction, vehicle (saline) or E . coli-LPS was injected at various doses (0.1, 1 and 5 mg/kg i.p.) for 7 days . The animals were sacrificed on day 8 after ulcer induction and the following parameters were analysed; ulcer area (planimetry), gastric blood flow (GBF) (H2 gas clearance method), gastric secretion, plasma levels of proinflammatory cytokines such as IL-1beta and TNFalpha, mucosal gene expression for cyclooxygenases (COX-1/-2), apoptosis-related proteins (Bax, Bcl-2), TNFalpha, IL-1beta and vascular endothelial growth factor (VEGF) . RESULTS: Daily parenteral challenge with LPS resulted in a dose-dependent delay in ulcer healing with maximum observed at a dose of 5 mg/kg (12.14 +/- 1.2 mm2 versus 5.18 +/- 0.8 mm2 in the control group) . The impairment of ulcer healing in LPS-treated rats was associated with a significant decrease in GBF, increased mRNA expression for IL-1beta, TNFalpha, the rise in plasma IL-1beta and TNFalpha levels, an overexpression of COX-2 and VEGF and imbalance in the ratio between pro-apoptotic Bax and antiapoptotic Bcl-2 . The daily administration of 50 mg/kg pentoxifylline by itself failed to accelerate the ulcer healing but attenuated the deleterious effects of LPS on this healing . CONCLUSIONS: 1) Bacterial endotoxin impairs ulcer healing through the decrease in gastric mucosal blood flow, increased expression and release of proinflammatory cytokines IL-1beta and TNFalpha, the imbalance between pro- and anti-apoptotic members of Bcl-2 family via downregulation of antiapoptotic bcl-2, and 2) endotoxin leads to upregulation of genes for VEGF and COX-2, which fail to accelerate the ulcer healing.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 1959 - 63
Fusobacterium equinum sp . nov., from the oral cavity of horses; Dorsch M et al.; Two strains of gram-negative, anaerobic, non-sporulating rod that were isolated from the normal oral cavity and oral-associated disease from horses and which phenotypically resembled Fusobacterium necrophorum were characterized by sequencing of the 16S rRNA gene, phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization . The results placed the novel strains as distinct members of the genus Fusobacterium . The novel species Fusobacterium equinum sp . nov . is proposed, with strain VPB 4027T (= NCTC 13176T = JCM 11174T) as the type strain.

Ann Ist Super Sanita, 2001, 37(2), 265 - 73
{Bacterial symbionts (Wolbachia) of filarial nematodes: implications for the treatment and pathology of filariasis}; Bazzocchi C et al.; Filarial nematodes harbour intracellular, Gram-negative bacteria belonging to the genus Wolbachia . These bacteria have been observed in various species of filariae, including the main filariasis agents of humans and animals . It has been suggested that Wolbachia could play an important role in the biology of filarial nematodes and could be implicated in the pathogenesis of filarial diseases . Wolbachia could thus represent a target for the control of filariasis and key to the understanding of these diseases . Indeed, in various species of filariae, tetracycline treatments have been shown both to reduce/eliminate the Wolbachia population and to determine detrimental effects on the nematodes . In addition, proteins of Wolbachia have been shown to determine specific IgG responses in animals infected by filariae and some Wolbachia molecules (e.g . LPS) have been shown to stimulate innate-immunity responses (e.g . production of cytokines such as IL1, IL6, IL10, TNF-alpha and IFN-gamma by macrophages).

J Mol Recognit, 2001 Nov-Dec, 14(6), 370 - 87
Towards a rational development of anti-endotoxin agents: novel approaches to sequestration of bacterial endotoxins with small molecules; David SA; Endotoxins, or lipopolysaccharides (LPS), present on the surface of Gram-negative bacteria, play a key role in the pathogenesis of septic shock, a common clinical problem and a leading cause of mortality in critically ill patients, for which no specific therapeutic modalities are available at the present time . The toxic moiety of LPS is a glycolipid called 'lipid A', which is composed of a bisphosphorylated diglucosamine backbone bearing up to seven acyl chains in ester and amide linkages . Lipid A is structurally highly conserved in Gram-negative bacteria, and is therefore an attractive target for developing anti-endotoxin molecules designed to sequester, and thereby neutralize, the deleterious effects of endotoxins . The anionic and amphipathic nature of lipid A enables the interaction of a wide variety of cationic amphiphiles with the toxin . This review describes the systematic evaluation of several structural classes of cationic amphiphiles, both peptides and non-peptidic small molecules, in the broader context of recent efforts aimed at developing novel anti-endotoxin strategies . The derivation of a pharmacophore for LPS recognition has led to the identification of novel, nontoxic, structurally simple small molecules, the lipopolyamines . The lipopolyamines bind and neutralize LPS in in vitro experiments as well as in animal models of endotoxicity, and thus present novel and exciting leads for rational, structure-based development of LPS-sequestering agents of potential clinical value .

Trends Microbiol, 2002 Jan, 10(1), 39 - 45
The E . coli alpha-hemolysin secretion system and its use in vaccine development; Gentschev I et al.; Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings . The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system . The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail . The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines . Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.

J Endotoxin Res, 2001, 7(6), 439 - 41
CD14 and LBP in endotoxemia and infections caused by Gram-negative bacteria; Heumann D; It is now well recognized that plasma LPS-binding protein (LBP) and membrane CD14 present at the surface of cells of the myelo/monocytic lineage are central molecules of the innate immune system, in response to LPS or to bacterial products . This paper reviews the role of LBP and CD14 in models of endotoxemia and infection.

Curr Opin Hematol, 2002 Jan, 9(1), 2 - 10
Toll-like receptors: how they work and what they do; Beutler B; The Toll-like receptors are the primary sensors of the innate immune system . This assignment of function was predicated on positional cloning, and was the result of long and painstaking inquiry into the mechanism of responses to bacterial endotoxin, the abundant lipopolysaccharide component of the outer membrane of Gram-negative bacteria . The sequence of events that led to the discovery of Toll-like receptor function carries an important lesson that should guide further analysis of the innate immune system.

Am J Respir Cell Mol Biol, 2002 Jan, 26(1), 152 - 9
Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology; Vernooy JH et al.; Lipopolysaccharide (LPS), a major proinflammatory glycolipid component of the gram-negative bacterial cell wall, is one of the agents ubiquitously present as contaminant on airborne particles, including air pollution, organic dusts, and cigarette smoke . Chronic exposure to significant levels of LPS is reported to be associated with the development and/or progression of many types of lung diseases, including asthma, chronic bronchitis, and progressive irreversible airflow obstruction, that are all characterized by chronic inflammatory processes in the lung . In the present study, pathologic effects of long-term LPS exposure to the lung were investigated in detail . To this end, a murine model in which mice were exposed to repeated intratracheal instillation of Escherichia coli LPS was developed . We show that long-term LPS instillation in mice results in persistent chronic pulmonary inflammation, characterized by peribronchial and perivascular lymphocytic aggregates (CD4(+), CD8(+), and CD19(+)), parenchymal accumulation of macrophages and CD8(+) T cells, and altered cytokine expression . Furthermore, airway and alveolar alterations such as mucus cell metaplasia, airway wall thickening, and irreversible alveolar enlargement accompanied the chronic inflammatory response . Interestingly, the observed inflammatory and pathologic changes mimic changes observed in human subjects with chronic inflammatory lung diseases, especially chronic obstructive pulmonary disease (COPD), suggesting that this murine model could be applicable to dissect the role of inflammation in the pathogenesis of these disease conditions.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 101 - 4
Single-dose intraperitoneal magainins improve survival in a gram-negative-pathogen septic shock rat model; Cirioni O et al.; The therapeutic efficacies of three polycationic peptides selected among the class of the magainins (magainin I, magainin II, and magainin II amide), alone and combined with piperacillin, were investigated in a rat model of septic shock . Rats were given an intraperitoneal injection of 2 x 10(10) CFU of Escherichia coli and randomized to receive intraperitoneally isotonic sodium chloride solution, 60 mg of piperacillin per kg of body weight, and 1 mg of each magainin per kg alone and combined with 60 mg of piperacillin per kg . The main outcome measures were bacterial growth in abdominal exudate and plasma, endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma, and lethality . Treatments with the magainins achieved significant reductions of bacterial growth and plasma endotoxin and TNF-alpha concentrations . In general, treatments with the combinations of magainins and piperacillin demonstrated the highest efficacies.

Ann Agric Environ Med, 2001, 8(2), 111 - 7
Use of mass spectrometry for characterising microbial communities in bioaerosols; Szponar B et al.; The use of chemical marker analysis for characterising microbial communities in organic dust samples is exemplified in a comparative study of dusts collected in a home and a swine confinement building, respectively . The chemical markers studied included 3-hydroxy fatty acids (markers of endotoxin), ergosterol (marker of fungal biomass), and muramic acid (marker of peptidoglycan/bacterial biomass) . Samples were hydrolysed and subjected to various chemical manipulations for rendering the markers suitable for gas chromatography-tandem mass spectrometry analysis . Considerable differences between the dust samples were revealed . Swine dust contained 46 ng/mg of ergosterol (house dust 2.1 ng/mg), 0.096 nmol/mg of endotoxin (house dust 0.020 nmol/mg), and 483 ng/mg of muramic acid (house dust 366 ng/mg) . The 3-hydroxy fatty acid and muramic acid results demonstrated a much higher proportion of Gram-negative bacteria to Gram-positives in swine dust than in house dust, and ergosterol results demonstrated a much higher proportion of fungi . The different distribution of 3-hydroxy fatty acids in the 2 samples illustrated differences in their flora of Gram-negative bacteria . The described method allows accurate determination of markers even when present down to trace levels in chemically complex matrices and should be useful in evaluating the role of microorganisms in the development of occupational lung disease, e.g . in agricultural environments.

J Biol Chem, 2002 Feb 15, 277(7), 5061 - 73 Epub 2001 Dec 06.
Groups IV, V, and X phospholipases A2s in human neutrophils: role in eicosanoid production and gram-negative bacterial phospholipid hydrolysis; Degousee N et al.; The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils . We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable . GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules . Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2) . GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum . By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

AAPS PharmSci . 1999;1(2):E2.
The Venus flytrap of periplasmic binding proteins: an ancient protein module present in multiple drug receptors; Felder CB et al.; Located between the inner and outer membranes of Gram-negative bacteria, periplasmic binding proteins (PBPs) scavenge or sense diverse nutrients in the environment by coupling to transporters or chemotaxis receptors in the inner membrane . Their three-dimensional structures have been deduced in atomic detail with the use of X-ray crystallography, both in the free and liganded state . PBPs consist of two large lobes that close around the bound ligand, resembling a Venus flytrap . This architecture is reiterated in transcriptional regulators, such as the lac repressors . In the process of evolution, genes encoding the PBPs have fused with genes for integral membrane proteins . Thus, diverse mammalian receptors contain extracellular ligand binding domains that are homologous to the PBPs; these include glutamate/glycine-gated ion channels such as the NMDA receptor, G protein-coupled receptors, including metabotropic glutamate, GABA-B, calcium sensing, and pheromone receptors, and atrial natriuretic peptide-guanylate cyclase receptors . Many of these receptors are promising drug targets . On the basis of homology to PBPs and a recently resolved crystal structure of the extracellular binding domain of a glutamate receptor ion channel, it is possible to construct three-dimensional models of their ligand binding domains . Together with the extensive information available on the mechanism of ligand binding to PBPs, such models can serve as a guide in drug discovery.

J Infect Dis, 2001 Dec 15, 184(12), 1524 - 31 Epub 2001 Dec 03.
Morphine-induced degradation of the host defense barrier: role of macrophage injury; Bhaskaran M et al.; The effect of morphine on the degradation of the host defense barrier in rats and mice was studied . Mice received either 3 or 11 doses of morphine . Mice receiving 11 doses of morphine showed gram-negative bacteremia and bacterial growth in samples of peritoneal fluid (PF), liver, spleen, kidneys, heart, and lungs; PF and tissue samples from only 1 control mouse showed bacterial growth, and no control mice had bacteremia . Mice receiving 11 doses also had suppressed bone marrow macrophage colony formation . Monocytes and peritoneal macrophages harvested from morphine-treated mice showed greater injury than did those from control mice . Pretreatment of mice with naloxone inhibited morphine-induced macrophage injury and degradation of the host defense barrier . In in vitro studies, morphine attenuated the killing of bacteria phagocytosed by macrophages and also facilitated their escape . This study indicates that morphine-induced monocyte and macrophage injury may be linked to degradation of the host defense barrier.

Eur J Biochem, 2001 Dec, 268(24), 6458 - 64
Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae; Jarvinen N et al.; Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer . The difference in clinical outcome has been suggested to be due to strain differences . H . pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions . The O-antigen of H . pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium . These Lewis glycans of H . pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium . In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor . Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H . pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae . The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.

J Surg Res, 2001 Dec, 101(2), 138 - 45
Bacterial wall products induce downregulation of vascular endothelial growth factor receptors on endothelial cells via a CD14-dependent mechanism: implications for surgical wound healing; Power C et al.; INTRODUCTION: Vascular endothelial growth factor (VEGF) is a potent mitogenic cytokine which has been identified as the principal polypeptide growth factor influencing endothelial cell (EC) migration and proliferation . Ordered progression of these two processes is an absolute prerequisite for initiating and maintaining the proliferative phase of wound healing . The response of ECs to circulating VEGF is determined by, and directly proportional to, the functional expression of VEGF receptors (KDR/Flt-1) on the EC surface membrane . Systemic sepsis and wound contamination due to bacterial infection are associated with significant retardation of the proliferative phase of wound repair . The effects of the Gram-negative bacterial wall components lipopolysaccharide (LPS) and bacterial lipoprotein (BLP) on VEGF receptor function and expression are unknown and may represent an important biological mechanism predisposing to delayed wound healing in the presence of localized or systemic sepsis . MATERIALS AND METHODS: We designed a series of in vitro experiments investigating this phenomenon and its potential implications for infective wound repair . VEGF receptor density on ECs in the presence of LPS and BLP was assessed using flow cytometry . These parameters were assessed in hypoxic conditions as well as in normoxia . The contribution of CD14 was evaluated using recombinant human (rh) CD14 . EC proliferation in response to VEGF was quantified in the presence and absence of LPS and BLP . RESULTS: Flow cytometric analysis revealed that LPS and BLP have profoundly repressive effects on VEGF receptor density in normoxic and, more pertinently, hypoxic conditions . The observed downregulation of constitutive and inducible VEGF receptor expression on ECs was not due to any directly cytotoxic effect of LPS and BLP on ECs, as measured by cell viability and apoptosis assays . We identified a pivotal role for soluble/serum CD14, a highly specific bacterial wall product receptor, in mediating these effects . The decreased VEGF receptor density on ECs accruing from the presence of bacterial wall products resulted in EC hyporesponsiveness to rhVEGF and significant abolition of VEGF-directed EC proliferation . CONCLUSION: These findings suggest that the well-recognized relationship between bacterial sepsis and attenuated wound healing may be due, in part, to the directly suppressive effects of bacterial wall components on EC VEGF receptor expression and, consequently, EC proliferation.

Pediatr Infect Dis J, 2001 Sep, 20(9), 914 - 5
Fatal sepsis associated with acute pancreatitis caused by Moraxella catarrhalis in a child; Ohkusu K et al.; We describe a 4-year-old boy with Cornelia de Lange syndrome who died of septic shock caused by Moraxella catarrhalis bacteremia . At autopsy there was evidence of acute hemorrhagic pancreatitis with abscesses . Gram-negative diplococci were seen histologically in the abscesses and pancreatic ducts.

J Oral Sci, 2001 Sep, 43(3), 159 - 63
Inhibition of hemolysis by antibody against the Porphyromonas gingivalis 130-kDa hemagglutinin domain; Abiko Y et al.; Porphyromonas gingivalis is a gram-negative anaerobic bacterial species implicated as an important pathogen in the development of adult periodontitis . Hemagglutinin may mediate the adsorption and invasion of bacteria into host cells . Furthermore, the hemagglutinin plays a role in the agglutinate and lyse erythrocytes intake of heme which is an absolute requirement for this bacterial growth . We previously cloned the gene encoding the 130-kDa hemagglutinin protein domain (130-kDa HMGD) and identified the functional motifs of agglutination of erythrocytes . Bacterial cell attachment to erythrocytes is an important initial step in the expression of hemolytic activity . In this study, we highly purified recombinant r130-kDa HMGD and prepared the specific antiserum . Further, the effect of the antibody on the hemolytic activity of P . gingivalis cells was examined . The polyclonal antibody recognized 43,49-kDa major bands in P . gingivalis cells and r130-kDa HMGD, and significantly inhibited the hemagglutinating and hemolytic activities of P . gingivalis cells . The findings suggest that the antibody may be useful in the development of the passive immunization against periodontal diseases caused by P . gingivalis infection.

Trends Microbiol, 2001 Dec, 9(12), 586 - 90
The role of bacterial pili in protein and DNA translocation; Koebnik R; Gram-negative bacteria have surface appendages that assemble via different secretion machineries . Recently, new experimental approaches have contributed to a better understanding of the molecular mechanisms of flagellar and pilus assembly, and protein secretion . These findings can be applied to plant pathogenic bacteria, which probably transfer effector proteins directly into their eukaryotic host cells . Here, it is suggested that assembly of Hrp pili occurs in the periplasm and that unfolded effector proteins attach to pilins within the pili, thus effecting protein translocation . A two-domain structure for the HrpA pilin from Pseudomonas syringae is also predicted.

Trends Microbiol, 2001 Dec, 9(12), 573 - 8
Polymorphic proteins of Chlamydia spp.--autotransporters beyond the Proteobacteria; Henderson IR et al.; Gram-negative bacteria secrete a variety of proteins to the cell surface and beyond, a process with many inherent difficulties . An exceptionally widespread answer to these problems is the type V (or autotransporter) secretion pathway . By exploiting the data made available by bacterial genome sequencing, we have discovered that the previously described polymorphic proteins of Chlamydia spp . resemble members of the autotransporter family, and we suggest that they follow the same secretion pathway.

Expert Opin Biol Ther, 2001 Sep, 1(5), 765 - 71
Bacterial ghosts as carrier and targeting systems; Lubitz W; Bacterial ghosts are empty cell envelopes originating from Gram-negative bacteria . They have a natural outer surface make-up which provides them with the original targeting functions of the bacteria they are derived from and are thus able to bind to and/or are taken up by specific cells or tissues of animal, human or plant origin . The extended bacterial ghost system represents a platform technology for creating new qualities in non-living carriers which can be used for the specific targeting of drugs, DNA or other compounds to overcome toxic or non-desired obstacles . Freeze dried bacterial ghosts are stable without the requirement of a cold chain and can be effectively administered orally and aerogenically as drug carriers . The new system is an alternative to liposomes and may have an advantage due to its higher specificity for targeting specific tissues, its easy method of production and its versatility in entrapping and packaging various compounds in different compartments of the carriers.

Int J Med Microbiol, 2001 Nov, 291(5), 331 - 43
Pathogenicity of Legionella pneumophila; Cianciotto NP; The bacterium Legionella pneumophila is the principal etiologic agent of Legionnaires' disease, a form of lobar pneumonia . Ubiquitous in aquatic environments, the gram-negative Legionella organism is a facultative, intracellular parasite of protozoa . The pathogenesis of legionellosis is largely due to the ability of L . pneumophila to invade and grow within alveolar macrophages, and it is widely believed that this ability results from a prior adaptation to intracellular niches in nature . Indeed, intracellular legionellae display a remarkable capacity to avoid endosomal and lysosomal bactericidal activities and to establish a unique replicative phagosome . In recent years, much progress has been made toward identifying the bacterial factors that promote intracellular infection and virulence . Surface structures that enhance infection include LPS, flagella, type IV pili, an outer membrane porin, and the Mip propyl-proline isomerase . Both type II and type IV protein secretion systems are critical for L . pneumophila pathogenesis . Whereas the type II (Lsp) system secretes a collection of degradative enzymes, the type IV (Dot/Icm) system likely exports effector proteins that are especially critical for trafficking of the Legionella phagosome . In addition to facilitating pilus formation and type II secretion, the inner membrane prepilin peptidase (PilD) of L . pneumophila appears to mediate a third, potentially novel pathway that is operative in the mammalian host . Periplasmic and cytosolic infectivity determinants include a catalase-peroxidase and the HtrA and Hsp60 stress-response proteins . The stationary phase response and the iron acquisition functions of L . pneumophila also play key roles in pathogenesis, as do a number of other loci, including the pts, mil and enh genes.

J Int Med Res, 2001 Sep-Oct, 29(5), 409 - 20
Bacterial lipopolysaccharide induces transforming growth factor beta and hepatocyte growth factor through toll-like receptor 2 in cultured human colon cancer cells; Yoshioka T et al.; This study examined, in human cancer lines, the pattern of cytokine production stimulated by lipopolysaccharide (LPS), a major component of outer surface of gram-negative bacteria, and characterized the expression pattern of CD14, cell surface LPS receptor antigen, and toll-like receptors (TLRs), which appear to be key regulators of the innate immune response system . Two colon cancer cell lines (DLD and LoVo), a hepatocellular carcinoma cell line and a myelomonocytic cell line were incubated with LPS for 0-72 h, and transforming growth factor (TGF) beta1 and beta2, hepatocyte growth factor (HGF) and interleukins 6, 8 and 15 were assayed . The only changes induced by incubation with LPS were significant increases in TGFbeta1 production at 12 h, and in HGF production at 72 h, in LPS-stimulated DLD cells, and significant increases in TGFbeta2 production after 12 h and in HGF after 72 h in LoVo cells . Using reverse transcriptase-polymerase chain reaction analysis, expression of CD14 and TLR-2 mRNA was detected in DLD and LoVo cells, and expression of TLR-4 mRNA was detected in PLC/PRF/5 and KG-1 cells . These results suggest that LPS induces TGFbeta and HGF production mediated by CD14/TLR-2 in cultured human colon cancer cell lines.

J Clin Microbiol, 2001 Dec, 39(12), 4568 - 70
Recurrent soft tissue abscesses caused by Legionella cincinnatiensis; Gubler JG et al.; Recurrent soft tissue abscesses of the jaw, wrist, and arm developed in a 73-year-old housewife with nephrotic syndrome and immunoglobulin A(kappa) gammopathy of unknown etiology . Conventional cultures remained negative, despite visible gram-negative rods on microscopy . Broad-spectrum PCR revealed Legionella cincinnatiensis, which was confirmed by isolation of the organism on special Legionella medium . Infections due to Legionella species outside the lungs are rare . L . cincinnatiensis has been implicated in only four cases of clinical infection; these involved the lungs in three patients and the central nervous system in one patient . We conclude that broad-spectrum PCR can be a valuable tool for the evaluation of culture-negative infections with a high probability of bacterial origin and that Legionella might be an underdiagnosed cause of pyogenic soft tissue infection.

Appl Environ Microbiol, 2001 Dec, 67(12), 5585 - 92
Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macrofaunal burrow sediments and description of Lutibacterium anuloederans gen . nov., sp . nov., and Cycloclasticus spirillensus sp . nov; Chung WK et al.; Two new polyaromatic hydrocarbon-degrading marine bacteria have been isolated from burrow wall sediments of benthic macrofauna by using enrichments on phenanthrene . Strain LC8 (from a polychaete) and strain M4-6 (from a mollusc) are aerobic and gram negative and require sodium chloride (>1%) for growth . Both strains can use 2- and 3-ring polycyclic aromatic hydrocarbons as their sole carbon and energy sources, but they are nutritionally versatile . Physiological and phylogenetic analyses based on 16S ribosomal DNA sequences suggest that strain M4-6 belongs to the genus Cycloclasticus and represents a new species, Cycloclasticus spirillensus sp . nov . Strain LC8 appears to represent a new genus and species, Lutibacterium anuloederans gen . nov., sp . nov., within the Sphingomonadaceae . However, when inoculated into sediment slurries with or without exogenous phenanthrene, only L . anuloederans appeared to sustain a significant phenanthrene uptake potential throughout a 35-day incubation . In addition, only L . anuloederans appeared to enhance phenanthrene degradation in heavily contaminated sediment from Little Mystic Cove, Boston Harbor, Boston, Mass.

Mol Microbiol, 2001 Nov, 42(3), 795 - 807
The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB; Cascales E et al.; The Tol-Pal system of Escherichia coli is required for the maintenance of outer membrane stability . Recently, proton motive force (pmf) has been found to be necessary for the co-precipitation of the outer membrane lipoprotein Pal with the inner membrane TolA protein, indicating that the Tol-Pal system forms a transmembrane link in which TolA is energized . In this study, we show that both TolQ and TolR proteins are essential for the TolA-Pal interaction . A point mutation within the third transmembrane (TM) segment of TolQ was found to affect the TolA-Pal interaction strongly, whereas suppressor mutations within the TM segment of TolR restored this interaction . Modifying the Asp residue within the TM region of TolR indicated that an acidic residue was important for the pmf-dependent interaction of TolA with Pal and outer membrane stabilization . Analysis of sequence alignments of TolQ and TolR homologues from numerous Gram-negative bacterial genomes, together with analyses of the different tolQ-tolR mutants, revealed that the TM domains of TolQ and TolR present structural and functional homologies not only to ExbB and ExbD of the TonB system but also with MotA and MotB of the flagellar motor . The function of these three systems, as ion potential-driven molecular motors, is discussed

Mol Biotechnol, 2001 Nov, 19(3), 279 - 96
The biology of endotoxin; Heine H et al.; Endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of Gram-negative bacteria and has profound immunostimulatory and inflammatory capacity . The septic shock syndrome caused by endotoxin still has an unacceptably high mortality rate and, owing to increasing numbers of resistant strains, remains an ongoing threat throughout the world . However, the past years have provided new insights especially into the receptors of the innate immune system that are involved into the recognition of LPS and the initial signal transduction pathways that are engaged after the primary recognition on the cell surface . The knowledge about the molecular basis for the responses to endotoxin may eventually lead to the development of new drugs to fight the fatal effects of bacterial infections.

FEMS Immunol Med Microbiol, 2001 Oct, 31(3), 169 - 73
Bactericidal activity of normal cord serum (NCS) against Gram-negative rods with sialic acid-containing lipopolysaccharides (LPS); Mielnik G et al.; Sialic acids, that are important constituents of animal tissue glycoconjugates, are also present in antigens of some bacterial strains . Capsular polysaccharides with sialic acid have been extensively studied whereas little is known on lipopolysaccharides which contain sialic acid . The susceptibility of Gram-negative strains with sialic acid-containing lipopolysaccharides to the bactericidal action of the sera of newborns was examined . The strains investigated showed variable sensitivity to the bactericidal action of normal cord serum.

J Endotoxin Res, 2001, 7(4), 315 - 21
Dual roles for HMGB1: DNA binding and cytokine; Czura CJ et al.; Effective therapies against overwhelming Gram-negative bacteremia, or sepsis, have eluded successful development . The discovery that tumor necrosis factor (TNF), a host-derived inflammatory mediator, was both necessary and sufficient to recapitulate Gram-negative sepsis raised cautious optimism for developing a targeted therapeutic . However, the rapid kinetics of the TNF response to infection defined an extremely narrow window of opportunity during which anti-TNF therapeutics could be successfully administered . HMGB1 was previously studied as a DNA-binding protein involved in DNA replication, repair, and transcription; and as a membrane-associated protein that mediates neurite outgrowth . A decade-long search has culminated in our identification of HMGB1 as a late mediator of endotoxemia . HMGB1 is released by macrophages upon exposure to endotoxin, activates many other pro-inflammatory mediators, and is lethal to otherwise healthy animals . Elevated levels of HMGB1 are observed in the serum of patients with sepsis, and the highest levels were found in those patients that died . The delayed kinetics of HMGB1 release indicate that it may be useful to target this toxic cytokine in the development of future therapies.

J Endotoxin Res, 2001, 7(4), 263 - 70
Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II; Gronow S et al.; L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria . In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd(2)-LPS, respectively . It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated . In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E . coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-beta-D-manno-heptopyranose . This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

Acta Crystallogr D Biol Crystallogr, 2001 Dec, 57(Pt 12), 1945 - 9 Epub 2001 Nov 21.
Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI; Watson WT et al.; Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria . AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein . The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4(3), with unit-cell parameters a = b = 66.40, c = 47.33 A . The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal . The rhenium-containing structure has been refined to a resolution of 2.5 A and the perrhenate ion binding sites and liganding residues have been identified.

J Feline Med Surg, 2000 Mar, 2(1), 19 - 27
Gastric helicobacters in cats; Lecoindre P et al.; The types of helicobacter which are found in the stomachs of carnivorous pets, especially cats, have been traditionally referred to as 'gastric helicobacter-like organisms' (GHLOs) . These are microaerophilic, Gram-negative, spiral bacteria with multiple terminal flagellae and are endowed with high-level urease activity which allows them to survive in an acidic environment . Certain species have one or more periplasmic fibrils . The two GHLOs most commonly found in cats are Helicobacter felis and a species related to H heilmannii which was recently cultured from dogs . All phenotypic and genotypic (16S RNA gene sequences) evidence suggests that both of these bacteria belong in the genus Helicobacter . Whether or not helicobacters can be transmitted to humans from carnivorous pets is controversial but the recent discovery of H pylori -infected cats may be evidence of an animal reservoir for this pathogen . Although the role of H pylori in inducing antral gastritis and perpetuating pyloric ulcers in humans is well established, whether or not Helicobacter spp are causally involved in any feline gastric inflammatory conditions is unknown .

Biomacromolecules, 2000 Summer, 1(2), 194 - 201
Identification of a marine benthic P(3HB)-degrading bacterium isolate and characterization of its P(3HB) depolymerase; Kasuya K et al.; A poly{(R)-3-hydroxybutyrate} (P(3HB))-degrading marine bacterium (strain NK-1, JCM10458) was isolated from the Pacific Ocean deep-sea floor (1165 m in depth) in Japan . The organism was a motile and Gram negative, aerobic, and rod-shaped bacterium, and its DNA had a guanine-plus-cytosine content of 57.7 mol% . On the basis of several phenotypic characters and a phylogenetic analysis of the gene coding for 16S rRNA, this strain was identified as Marinobacter sp . The strain required sodium salt for growth in the medium and secreted a P(3HB) depolymerase into the supernatant when it was cultivated on (S)-3-hydroxybutyric acid or P(3HB) as the sole carbon source . The P(3HB) depolymerase (PhaZMsp) was purified to homogeneity from the culture supernatant of Marinobacter sp . by hydrophobic and ion exchange column chromatography and showed a molecular mass of 70 kDa . PhaZMsp was stable at temperatures below 37 degrees C and at pH values of 7.5-10.0 . The N-terminal amino acid sequences of both the purified enzyme and the truncated one shared high homologies to the N-terminal and internal sequences of Pseudomonas stutzeri depolymerase, respectively . High-performance liquid chromatography analysis revealed that the enzymatic products of P(3HB) yielded monomer, dimer, and trimer of 3-hydroxybutyric acid . PhaZMsp was capable of hydrolyzing P(3HB), poly(3-hydroxypropionate), and poly(4-hydroxybutyrate).

Transplantation, 2001 Nov 15, 72(9), 1575 - 7
Microbiologic investigation on patients with cystic fibrosis subjected to bilateral lung transplantation; Trancassini M et al.; BACKGROUND: In cystic fibrosis (CF) patients, lung transplantation is the only way to improve both quality and length of life . Data in the literature show that, in 80% of the cases, mortality after lung transplantation in CF patients is due to infections . METHODS: We microbiologically monitored 34 patients subjected to bilateral lung transplantation in during 1996 to 1999 to ascertain whether a change in the bacterial species isolated from the lower respiratory tract took place that might have influenced the clinical conditions of the patients . RESULTS: Our results show that the percentage of nonfermenting Gram-negative bacteria isolated from the lower respiratory tract remains high even in the posttransplantation phase . Nevertheless, the general clinical conditions of most of the patients were good and the three patients who died did not do as a consequence of an infection . CONCLUSIONS: Lung transplantation constitutes a valid therapeutic choice for CF patients because the microorganisms that we isolated from the lungs of the patients in our study behave mostly as contaminants rather than as colonizers . However, the transplanted patients remain at risk and thus require constant microbiological surveillance.

Mol Microbiol, 2001 Oct, 42(2), 395 - 413
FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division; Chen JC et al.; During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials . In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA . All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site . We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK . We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra . Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN . These localization results support the model that E . coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.

Mol Microbiol, 2001 Oct, 42(2), 369 - 82
Regulation of the HpyII restriction-modification system of Helicobacter pylori by gene deletion and horizontal reconstitution; Aras RA et al.; Helicobacter pylori, Gram-negative, curved bacteria colonizing the human stomach, possess strain-specific complements of functional restriction-modification (R-M) systems . Restriction-modification systems have been identified in most bacterial species studied and are believed to have evolved to protect the host genome from invasion by foreign DNA . The large number of R-Ms homologous to those in other bacterial species and their strain-specificity suggest that H . pylori may have horizontally acquired these genes . A type IIs restriction-modification system, hpyIIRM, was active in two out of the six H . pylori strains studied . We demonstrate now that in most strains lacking M.HpyII function, there is complete absence of the R-M system . Direct DNA repeats of 80 bp flanking the hpyIIRM system allow its deletion, resulting in an "empty-site" genotype . We show that strains possessing this empty-site genotype and strains with a full but inactive hpyIIRM can reacquire the hpyIIRM cassette and functional activity through natural transformation by DNA from the parental R-M+ strain . Identical isolates divergent for the presence of an active HpyII R-M pose different restriction barriers to transformation by foreign DNA . That H . pylori can lose HpyII R-M function through deletion or mutation, and can horizontally reacquire the hpyIIRM cassette, is, in composite, a novel mechanism for R-M regulation, supporting the general hypothesis that H . pylori populations use mutation and transformation to regulate gene function.

J Biol Chem, 2002 Jan 4, 277(1), 618 - 22 Epub 2001 Nov 07.
Megalin deficiency offers protection from renal aminoglycoside accumulation; Schmitz C et al.; Aminoglycosides are antibiotics commonly used to treat life-threatening Gram-negative bacterial infections . However, their use is hampered by their severe nephrotoxicity due to accumulation in renal proximal tubules . Several pathways have been implicated in the renal uptake of aminoglycosides including megalin, an endocytic receptor in proximal tubular cells . Here, we have used mouse models with genetic or functional megalin deficiency to explore the contribution of megalin and other pathways to renal aminoglycoside uptake in vivo . We demonstrate that the uptake of aminoglycosides into the kidney directly correlates with renal megalin activity and is completely eliminated in mice lacking the receptor . Thus, our studies provide unequivocal evidence that megalin is the only major pathway responsible for renal aminoglycoside accumulation and that the receptor represents a unique drug target to prevent aminoglycoside-induced nephrotoxicity in patients.

Annu Rev Genet, 2001, 35, 439 - 68
Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing; Fuqua C et al.; Quorum sensing is an example of community behavior prevalent among diverse bacterial species . The term "quorum sensing" describes the ability of a microorganism to perceive and respond to microbial population density, usually relying on the production and subsequent response to diffusible signal molecules . A significant number of gram-negative bacteria produce acylated homoserine lactones (acyl-HSLs) as signal molecules that function in quorum sensing . Bacteria that produce acyl-HSLs can respond to the local concentration of the signaling molecules, and high population densities foster the accumulation of inducing levels of acyl-HSLs . Depending upon the bacterial species, the physiological processes regulated by quorum sensing are extremely diverse, ranging from bioluminescence to swarming motility . Acyl-HSL quorum sensing has become a paradigm for intercellular signaling mechanisms . A flurry of research over the past decade has led to significant understanding of many aspects of quorum sensing including the synthesis of acyl-HSLs, the receptors that recognize the acyl-HSL signal and transduce this information to the level of gene expression, and the interaction of these receptors with the transcriptional machinery . Recent studies have begun to integrate acyl-HSL quorum sensing into global regulatory networks and establish its role in developing and maintaining the structure of bacterial communities.

Extremophiles, 2001 Oct, 5(5), 343 - 9
Assignment of Pseudomonas sp . strain E-3 to Pseudomonas psychrophila sp . nov., a new facultatively psychrophilic bacterium; Yumoto I et al.; A facultatively psychrophilic bacterium, previously described as Pseudomonas sp . strain E-3, has been reassigned by phenotypic characterization, chemotaxonomic analysis, DNA-DNA hybridization, and 16S rRNA gene phylogenetic analysis . The organism was a gram-negative, aerobic . straight rod with polar flagella . It was catalase positive and oxidase positive, able to grow at -1 degree C but not at 40 degree C, and produced acid from D-glucose under aerobic conditions . The major isoprenoid quinone was ubiquinone-9, and the DNA G + C content was 57.2 mol% . Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacterium is a member of the genus Pseudomonas and was closest to Pseudomonas fragi . Determination of the DNA-DNA relatedness between strain E-3 and P . fragi revealed too low a level of homology (47.9%-51.3%) to identify them as the same species . On the basis of phenotypic characteristics, phylogenetic analysis, and DNA-DNA relatedness data, it is concluded that strain E-3 represents an individual species . Accordingly, the name Pseudomonas psychrophila is proposed . The type strain is E-3T (= JCM 10889).

Shock, 2001 Nov, 16(5), 334 - 9
Inflammatory response during abdominal and thyroid surgery: a prospective clinical trial on mediator release; Bolke E et al.; Several studies have been demonstrated that endotoxin is a potent stimulus of the acute inflammatory response following traumatic injury . Although numerous studies have indicated that the extent of surgical intervention correlates well with the inflammatory response, the potential role of endotoxin as a trigger under those conditions still remains unknown . Therefore, the aim of this study was to elucidate whether or not the up-regulated inflammatory mediators are paralleled by increased endotoxin plasma levels during and following surgery, and whether the extent of surgical intervention represents a crucial factor under those conditions . To study this, plasma was collected at various time points during and after surgery from 52 patients subjected to abdominal surgery (i.e., major surgery) and 25 patients subjected to thyroid surgery (i.e., minor surgery) . Plasma was assessed for endotoxin, endotoxin neutralizing capacity (ENC), and inflammatory mediators (leucotriene-C4 {LTC4}-, 6-keto-prostaglandin-F-1-alpha {PGF}-, thromboxane-B2 {TxB2}, interleukin-6 {IL-6}, and C-reactive protein {CRP}) . Furthermore, splanchnic blood circulation was measured by determination of the intraluminal pH of the stomach and sigma (pHi) by intraluminal tonometry . Mesenteric lymph nodes were also collected at the time point of organ mobilization in the major surgery group and were assessed for bacterial translocation . Among all parameters investigated, endotoxin showed the most rapid changes . A significant increase in plasma levels of endotoxin and a decrease of ENC were found in the major surgery groups following induction of anesthesia and in the minor surgery groups after skin incision . Moreover, the incidence of elevated endotoxin levels was significantly higher (89% with elevated endotoxin levels) than the incidence of bacterial translocation (35% with gram-negative bacteria) in mesenterial lymph nodes of the major surgery group . pHi decreased significantly in both groups after skin incision, but no difference was observed between the major and minor surgery groups . Plasma mediators of the arachidonic acid cascade (LTC4, PGF, and TxB2) were only elevated in individual patients during and following surgery in both groups . Conversely, the post-operative increase in the acute phase mediators was significantly different in the major and minor surgery groups . IL-6 plasma levels peaked higher and earlier after major surgery than after minor surgery and the delayed increase of CRP was significantly greater in the major surgery group . In conclusion, the results indicate that plasma levels of endotoxin significantly correlate with the severity of the surgical intervention and may play an important role in inducing mediators of the acute phase reaction under such conditions.

J Bacteriol, 2001 Dec, 183(23), 6733 - 9
Analyses of mrp genes during Myxococcus xanthus development; Sun H et al.; Myxococcus xanthus is a gram-negative soil bacterium that undergoes development under starvation conditions . Our previous study identified a new genetic locus, mrp, which is required for both fruiting body formation and sporulation . The locus encodes two transcripts: mrpAB, which consists of a histidine kinase and an NtrC-like response regulator, and mrpC, a cyclic AMP receptor protein family transcription activator . In this study, we used genetic and biochemical analyses to investigate the possible interactions between the mrp genes and other known developmental genes and events . These studies show that the mrp genes possibly function after A-signaling and (p)ppGpp but before C-signaling and that they regulate various early and late developmental genes and events.

J Mol Biol, 2001 Nov 2, 313(4), 785 - 95
A minor capsid protein P30 is essential for bacteriophage PRD1 capsid assembly; Rydman PS et al.; Bacteriophage PRD1 is a double-stranded DNA virus infecting Gram-negative hosts . It has a membrane component located in the interior of the isometric capsid . In addition to the major capsid protein P3, the capsid contains a 9 kDa protein P30 . Protein P30 is proposed to be located between the adjacent facets of the icosahedral capsid and is required for stable capsid assembly . In its absence, an empty phage-specific membrane vesicle is formed . The major protein component of this vesicle is a phage-encoded assembly factor, protein P10, that is not present in the final structure .

Prog Lipid Res, 2002 Jan, 41(1), 27 - 65
Human immunodeficiency virus and host cell lipids . Interesting pathways in research for a new HIV therapy; Raulin J; It has been reported in the literature that biological membranes arising from HIV-induced cell fusion, as well as syncytium formation between infected and non-infected cells and those involved in transduction, viral DNA nuclear import and virion budding from the host cell, are all made of proteins, a phospholipid (P) bilayer and cholesterol (C) . However, the P/C molar ratio is higher in the retroviral envelope than in the plasma membrane where they originate, and higher than in the nuclear envelope . Mechanisms are described which elucidate this puzzling fact, as well as cholesterol-dependent leakage and pore formation during cell fusion . Fatty acylation of viral and host cell proteins is required to direct them to membranes . Detergent-insoluble microdomains enriched in cholesterol and sphingolipids, termed either DIGs (detergent-insoluble glycolipid-enriched complexes), DRMs (detergent resistant membranes), TIFFs (Triton-insoluble floating fractions) or GEMs (glycolipid-enriched membranes), function as platforms for attachment of proteins in the process of signal transduction . HIV-SUgp120 (HIV-surface glycoprotein), T-cell receptor (TCR)-CD4+ and co-receptors promote aggregation of these lipid "rafts" which concentrate the Src family tyrosine kinases SFKs (PTK, Lyn, Fyn, Lck), GPI (glycosyl phosphatidylinositol)-anchored proteins, and phosphatidylinositol kinases PI(3)K and PI(4)K, inducing cell signalling . HIV-SUgp120 transduces the activation signal and provokes the formation of polyunsaturated fatty acid (PUFA) metabolites, i.e . the prostaglandin PGE2 suppressor of immune function and inhibitor of cytotoxic T-lymphocyte (CTL) proliferation, while PGB2 activates SFKs and increases mRNA expression, as well as NFkappaB (nuclear transcription factor) translocation to nucleus . HIV nuclear import, DNA integration, chromatin template capacity may be mediated by the lipid environment . The lipid-enriched microdomains from which HIV-1 buds, may explain the high level of cholesterol and sphingolipids in the viral envelope, since host cell rafts become a viral coat . HIV-1 infection induces alteration of cellular lipids: (1) shift in phospholipid synthesis to neutral lipids associated with the viral load, polyunsaturated fatty acid (PUFA) peroxidation, and n-3 deficiency with deregulation of cytokines and PPAR-gamma (peroxisome proliferator-activated receptor-gamma), and (2) alloimmune phospholipid antibody production in which antibodies to cardiolipin and to phosphatidylserine are most prevalent, due to the destruction of mitochondrial membranes and progression of lymphocyte apoptosis . The current highly active anti-retroviral therapy, including both viral reverse transcriptase (RT) inhibitors (NRTIs and NNRTIs, nucleoside and non-nucleoside RT inhibitors) and protease inhibitors (PIs), induces side-effects in the long term . Lipodystrophy (LD), consists of peripheral lipoatrophy associated with central fat accumulation (called "crixbelly" and "buffalo hump"), insulin resistance, elevation of very low density lipoproteins, decrease in high density lipoproteins and inhibition of adipocyte differentiation . LD syndrome appears to be induced by PIs that inhibit GLUT4, glucose transporter isoform, and by NRTIs which provoke mitochondrial failure . New therapeutic strategies assessed: (1) inhibition of the viral integrase and/or HIV entry into cells through natural products or their derivatives, (2) inhibition of HIV-1 entry into macrophages pretreated with Gram-negative bacterial lipopolysaccharide, (3) vaccination with multi-lipopeptides, i.e . sequences of HIV-1 peptides with CD4+ T-cell and B-cell epitopes, modified by adding a lipid tail to one end, which produce HIV-specific CTL and multispecific immune responses in most of the vaccinated subjects and (4) stimulation of antiviral drug activity with lipid-prodrugs targeting viral RT, polymerase, integrase, or aspartyl-protease.

Front Biosci, 2001 Nov 01, 6, E168 - 86
Mechanisms of Bordetella pathogenesis; Mattoo S et al.; Bordetella are Gram negative bacteria that cause respiratory tract infections in humans and animals . While at least five different species of Bordetella are known to exist, this review focuses on B . pertussis, B . bronchiseptica and B . parapertussis subspecies . In their virulent phase, all of these bacteria produce a nearly identical set of virulence factors which include adhesins such as filamentous hemagglutinin (FHA), fimbriae and pertactin, as well as toxins such as a bifunctional adenylate cyclase/hemolysin, dermonecrotic toxin, tracheal cytotoxin, a B . pertussis specific pertussis toxin and B . bronchiseptica specific type III secreted proteins . Expression of nearly all of these virulence factors is positively regulated by the products of the bvgAS locus . BvgA and BvgS comprise a two-component signal transduction system that mediates transition between at least three identifiable phases---a virulent (Bvg+) phase, an avirulent (Bvg-) phase and an intermediate (Bvg(i)) phase---in response to specific environmental signals . Bordetella colonize the ciliated respiratory mucosa, a surface designed to eliminate foreign particles, thereby making the adherence and persistence mechanisms of these bacteria crucial . The development of relevant animal models for B . bronchiseptica has enabled us to study Bordetella pathogenesis in the context of natural host-pathogen interactions . In addition, evolutionary studies across the various Bordetella species and detailed analysis of differential regulation of Bvg-activated/repressed genes has greatly enhanced our understanding of the mechanisms of Bordetella pathogenesis.

Vutr Boles, 2000, 32(4), 33 - 40
{The role of bacterial endotoxins, receptors and cytokines in the pathogenesis of septic (endotoxin) shock}; Lazarov S et al.; Sepsis, resistant to therapy, results in the development of septic (endotoxin) shock . The latter is caused by the endotoxins of different Gram-negative bacteria . Endotoxin (bacterial lipopdisacharide--LPS) interacts with cells through specific membrane or plasma soluble endotoxin receptors (sCD14, mlD14, LBP, CD13/CD14, CD16, CD116/CD18, L-selectin, etc.) . Endotoxin interaction with the mCD14 receptor of the monocytes, macrophages and the neutrophils results in the production of a number of proinflammatory cytokines--tumor necrosis factor alpha (TNF alpha), interleukines 1 and 6 (IL-1 and IL-6, etc), antiinflammatory cytokines--interleukines 10 and 12 (IL-10 and IL-12), cell adhesion molecules (P-selectin, E-selectin, ICAM-1, VCAM-1, etc.) and inducible enzymes: inducible NO synthase (iNOS), inducible phospholipase A2 (cPL-A2), inducible cyclooxygenase (COX-2) . All pathologic processes in the structure and function of human body during endotoxin shock are a result of the disbalance of a number of mediators with a proinflammatory and antiinflammatory effects.

Vutr Boles, 2000, 32(4), 18 - 24
{The role of cell adhesion molecules and proinflammatory mediators in the pathogenesis of endotoxin adult respiratory distress syndrome}; Lazarov S et al.; The Gram-negative bacterial sepsis that is resistant to therapy results in the development of septic shock and the multiple organ dysfunction syndrome (MODS) . It is established that the adult respiratory distress syndrom (ARDS) as a component of MODS is the reason for high mortality in these patients . The basic pathogenetic link between ARDS and the septic shock are the lesions of the alveolo-capillary membrane . They result from the sequestrated neutrophil cells in the lungs . Neutrophil extravasation is manifested by the following steps (stages): 1) weak initial adhesion; 2) rolling; 3) stable adhesion; and 4) extravasation . These processes are mediated by cell adhesion molecules that belong to four classes: selectins, selectin ligands, integrins and immunoglobulin superfamily . Thus cell adhesion molecules mediate the sequestration of neutrophil cells in the lungs and the damage of the pulmonary surfactant system.

Arch Pharm (Weinheim), 2001 Sep, 334(8-9), 295 - 301
Conjoint molecules of cephalosporins and aminoglycosides; Grapsas I et al.; A general synthetic route to conjoint molecules of cephalosporins and aminoglycosides is described . These molecules were designed as potential substrates for bacterial beta-lactamases, enzymes that hydrolyze the beta-lactam bond of cephalosporins . Hydrolysis of the beta-lactam bond was expected to release the C10-appended aminoglycoside . Since beta-lactamases are sequestered in the periplasmic space of gram-negative bacteria, this sequence of events would liberate aminoglycoside inside such bacteria . It is expected that such local delivery of aminoglycosides would circumvent the inherent toxicity of aminoglycosides that occurs during systemic exposure within the mammalian host.

Invest Ophthalmol Vis Sci, 2001 Nov, 42(12), 2867 - 77
The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells; Song PI et al.; PURPOSE: Gram-negative bacterial infections of the eye can lead to corneal bacterial keratitis, visual impairment, and blindness . Many of these pathologic changes may be mediated by bacterially derived products such as lipopolysaccharide (LPS) . In this investigation, it has been established for the first time that human corneal cells are capable of expressing the functional LPS receptor complex proteins, CD14 and Toll-like receptor 4 (TLR4) . METHODS: CD14 and TLR4 mRNA expression in human corneal cells was determined by RT-PCR and Northern blot analysis, and cell surface expression of these proteins was measured by flow cytometry . LPS-mediated corneal cell activation was determined by measuring intracellular calcium mobilization . Cellular cytokine and chemokine secretion in response to LPS was measured by ELISA . The expression and localization of CD14 in whole human cornea was determined by immunohistochemistry . RESULTS: Human corneal epithelial, stromal, and endothelial cells expressed CD14 mRNA and cell surface CD14 . LPS binding to cornea CD14 resulted in a rapid intracellular calcium response and the secretion of multiple proinflammatory cytokines and chemokines . CD14 mRNA expression in corneal epithelial cells was upregulated by LPS . In addition to CD14, corneal epithelial cells expressed the functional LPS receptor-signaling protein TLR4, which was also augmented by LPS . CONCLUSIONS: The cornea expresses functional CD14 and TLR4 LPS receptor proteins . Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections.

Curr Microbiol, 2001 Dec, 43(6), 444 - 7
Microbial detection with low molecular weight RNA; Kourentzi KD et al.; The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA) . We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology . These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement . The assays can be used with both crude cell lysates and purified total RNA preparations . Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria . Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E . coli strain . Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Biochemistry, 2001 Nov 6, 40(44), 13177 - 87
Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes; Nguyen NT et al.; We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria . Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism . This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties . Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis . The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K . This latter loop is present only in type II citrate synthase sequences . Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network . Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer . The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases . This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation . Another surprising observation from the structure of type II E . coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site . Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur . Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure . Thus, acetylcoenzyme A binding to type II E . coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite . We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases . Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.

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