Proc Natl Acad Sci U S A . 2005 Jan 14; {Epub ahead of print} Imaging bacterial infections with radiolabeled 1-(2'deoxy-2'-fluoro-{beta}-D-arabinofuranosyl)-5-iodouracil; Bettegowda C et al.; Bacterial infections provide diagnostic dilemmas that could be enlightened by modern imaging technologies . We have developed a simple method for imaging bacterial infections in mice that relies on the phosphorylation and trapping of the thymidine kinase (TK) substrate 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-{(125)I}iodouracil ({(125)I}FIAU) within bacteria . FIAU was found to inhibit the growth of WT Escherichia coli but not a TK(-) strain, indicating that WT E . coli could metabolize this compound . In silico analyses demonstrated that all pathogenic strains of bacteria whose genomes have been sequenced contain a TK gene highly homologous to the E . coli TK . Accordingly, we demonstrated that localized infections caused by representatives of five genera of bacteria could be readily imaged with {(125)I}FIAU . Such imaging provides a general method for the diagnosis of localized bacterial infections that could be translatable to the clinic. Science, 2005 Jan 14, 307(5707), 254 - 8 CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance; Niess JH et al.; Dendritic cells (DCs) and macrophages are critical to innate and adaptive immunity to the intestinal bacterial microbiota . Here, we identify a myeloid-derived mucosal DC in mice, which populates the entire lamina propria of the small intestine . Lamina propria DCs were found to depend on the chemokine receptor CX3CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens . CX3CR1 was also found to control the clearance of entero-invasive pathogens by DCs . Thus, CX3CR1-dependent processes, which control host interactions of specialized DCs with commensal and pathogenic bacteria, may regulate immunological tolerance and inflammation. Zh Mikrobiol Epidemiol Immunobiol, 2004 Nov-Dec, (6), 106 - 13 {Role of apoptosis in the regulation of the infectious process}; Intimin-mediated export of passenger proteins requires maintenance of a translocation-competent conformation; Abteilung fur Molekulare Genetik und Praparative Molekularbiologie, Grisebachstr . 8, D-37077 Gottingen, Germany . HKolmar@Uni-MolGen.gwdg.deIntimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells . Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor . We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REI(v), ubiquitin, calmodulin, beta-lactamase inhibitor protein, or beta-lactamase . By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure . We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane . These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface. J Biochem (Tokyo), 2004 Oct, 136(4), 409 - 13 Cytolethal distending toxin: a bacterial bullet targeted to nucleus; Ohara M et al.; Cytolethal distending toxin (Cdt) is a newly added member of bacterial protein toxins that hijack the control system of eukaryotic cells . Cdts are produced by several pathogenic bacteria causing chronic infectious diseases . They are composed of three subunits, CdtA, CdtB and CdtC, which together form a ternary complex . CdtB is the active component, and CdtA and CdtC are involved in delivering the CdtB into the cells . The sophisticated strategy of Cdt to control host cells is CdtB-mediated limited DNA damage of the host cell chromosome, which triggers the response of the cell cycle checkpoint and results in G2 arrest in the cells . Cdt also induces apoptotic cell death of lymphocytes, which may be relevant to onset or persistence of chronic infection by the producing bacteria . The study of this toxin is expected to provide us information on a novel strategy by which bacteria interact with host cells. J Gen Virol, 2005 Jan, 86(Pt 1), 159 - 69 Genome sequence of the non-pathogenic strain 15 of pneumonia virus of mice and comparison with the genome of the pathogenic strain J3666; Thorpe LC et al.; Pneumonia virus of mice (PVM) is a member of the subfamily Pneumovirinae and is the closest known relative of respiratory syncytial virus . Both viruses cause pneumonia in their respective hosts . Here, the genome sequences of two strains of PVM, non-pathogenic strain 15 and pathogenic strain J3666, are reported . Comparison of the genome sequences revealed 59 nucleotide differences between the two strains, 37 of which were coding . The nucleotide differences were spread throughout the genome, affecting cis-acting regulatory regions and seven of the ten genes . Development of a reverse-genetics system for PVM should allow further elucidation of the functional importance of the genetic differences between the two strains identified here. Nippon Rinsho, 2004 Dec, 62(12), 2276 - 80 {Rapid diagnostic system of bacterial infection}; Ezaki T et al.; Approach to develop genetic detection system for bacterial infection in blood is a demanding subject because number of bacteria in blood stream is very few and in most cases the number of bacteria is less than the detection limit of gene amplification methods . At the first stage of blood sampling, we only applied gene amplification method against fastidious organisms to culture and organisms causes severe infection . Organisms which are usually found very few in blood are subjected to blood culture . After monitoring system of blood culture gave positive signal of bacterial growth, DNA or RNA in culture media were extracted and amplified with universal primers for bacteria and fungi . Amplicons were applied to a rapid sequencer . However, in case of mixed infection, the sequencing is not successful . In such cases, the amplicons are analized with DNA microarray, immobilized 16S rDNA and 28S rDNA of 1,000 human pathogenic bacteria and fungi. J Virol, 2005 Jan, 79(1), 428 - 40 Importance of arginine 20 of the swine vesicular disease virus 2A protease for activity and virulence; Inoue T et al.; A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease . The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs) . The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y . Sakoda, N . Ross-Smith, T . Inoue, and G . J . Belsham, J . Virol . 75:10643-10650, 2001) . Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed . Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued . The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity . In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected . Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred . The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease. J Med Microbiol, 2004 Dec, 53(Pt 12), 1221 - 7 The Bartonella henselae sucB gene encodes a dihydrolipoamide succinyltransferase protein reactive with sera from patients with cat-scratch disease; Litwin CM et al.; Bartonella henselae is a recently recognized pathogenic bacterium associated with cat-scratch disease, bacillary angiomatosis and bacillary peliosis . A recombinant clone expressing an immunoreactive antigen of B . henselae was isolated by screening a genomic DNA cosmid library by Western blotting with sera pooled from patients positive for B . henselae IgG antibodies by indirect immunofluorescence (IFA) . The deduced amino acid sequence of the 43.7 kDa encoded protein was found to be 76.3 % identical to the dihydrolipoamide succinyltransferase enzyme (SucB) of Brucella melitensis . SucB has been shown to be an immunogenic protein during infections by Brucella melitensis, Coxiella burnetii and Bartonella vinsonii . The agreement between reactivity with a recombinant SucB fusion protein on immunoblot analysis and the results obtained by IFA was 55 % for IFA-positive sera and 88 % for IFA-negative sera . Cross-reactivity was observed with sera from patients with antibodies against Brucella melitensis, Mycoplasma pneumoniae, Francisella tularensis, Coxiella burnetii and Rickettsia typhi. Proc Natl Acad Sci U S A, 2004 Dec 14, 101(50), 17389 - 93 Epub 2004 Dec 06. Catalysis of protein folding by chaperones in pathogenic bacteria; Bann JG et al.; Molecular chaperones are thought to inhibit off-pathway interactions such as aggregation from occurring without influencing the on-pathway formation of native structure . Here, we present a mechanism whereby the family of PapD-like chaperones, which are involved in the formation of adhesive pili in pathogenic bacteria, function by suppressing aggregation while simultaneously catalyzing the folding of subunits that make up the pilus . We also show that the Arg-8 residue, invariant in the cleft of all known PapD-like chaperones, makes up part of the active site of the chaperone . The data argue for a temporal mechanism of catalyzed folding . The terminal carboxylate group of a pilus subunit anchors to the active site of the chaperone by hydrogen bonding . This bonding spatially fixes the COOH terminus of the subunit in the correct context for beta-sheet formation, using the edge of the NH(2)-terminal domain of the chaperone as a nucleation site. Mol Phylogenet Evol, 2005 Jan, 34(1), 15 - 28 Bayesian phylogenetic analysis reveals two-domain topology of S-adenosylhomocysteine hydrolase protein sequences; Stepkowski T et al.; S-Adenosylhomocysteine hydrolase (SahH) is involved in the degradation of the compound which inhibits methylation reactions . Using a Bayesian approach and other methods, we reconstructed a phylogenetic tree of amino acid sequences of this protein originating from all three major domains of living organisms . The SahH sequences formed two major branches: one composed mainly of Archaea and the other of eukaryotes and majority of bacteria, clearly contradicting the three-domain topology shown by small subunit rRNA gene . This topology suggests the occurrence of lateral transfer of this gene between the domains . Poor resolution of eukaryotes and bacteria excluded an ultimate conclusion in which out of the two domains this gene appeared first, however, the congruence of the secondary branches with SS rRNA and/or concatenated ribosomal protein datasets phylogenies suggested an "early" acquisition by some bacterial and eukaryotic phyla . Similarly, the branching pattern of Archaea reflected the phylogenies shown by SS rRNA and ribosomal proteins . SahH is widespread in Eucarya, albeit, due to reductive evolution, it is missing in the intracellular parasite Encephalitozoon cuniculi . On the other hand, the lack of affinity to the sequences from the alpha-Proteobacteria and cyanobacteria excludes a possibility of its acquisition in the course of mitochondrial or chloroplast endosymbioses . Unlike Archaea, most bacteria carry MTA/SAH nucleosidase, an enzyme involved also in metabolism of methylthioadenosine . However, the double function of MTA/SAH nucleosidase may be a barrier to ensure the efficient degradation of S-adenosylhomocysteine, specially when the intensity of methylation processes is high . This would explain the presence of S-adenosylhomocysteine hydrolase in the bacteria that have more complex metabolism . On the other hand, majority of obligate pathogenic bacteria due to simpler metabolism rely entirely on MTA/SAH nucleosidase . This could explain the observed phenetic pattern in which bacteria with larger (>6 Mb-million base pairs) genomes carry SAH hydrolase, whereas bacteria that have undergone reductive evolution usually carry MTA/SAH nucleosidase . This suggests that the presence or acquisition of S-adenosylhomocysteine hydrolase in bacteria may predispose towards higher metabolic, and in consequence, higher genomic complexity . The good examples are the phototrophic bacteria all of which carry this gene, however, the SahH phylogeny shows lack of congruence with SSU rRNA and photosyntethic genes, implying that the acquisition was independent and presumably preceded the acquisition of photosyntethic genes . The majority of cyanobacteria acquired this gene from Archaea, however, in some species the sahH gene was replaced by a copy from the beta- or gamma-Proteobacteria. Plant Cell, 2004 Dec, 16(12), 3496 - 507 Epub 2004 Dec. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants; Kunze G et al.; Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs) . Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae . EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli . Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses . The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors . In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria. J Bacteriol, 2004 Dec, 186(23), 7936 - 43 In vivo activity of enzymatic and regulatory components of the phosphoenolpyruvate:sugar phosphotransferase system in Mycoplasma pneumoniae; Halbedel S et al.; Mycoplasma pneumoniae is a pathogenic bacterium that is highly adapted to life on mucosal surfaces . This adaptation is reflected by the very compact genome and the small number of regulatory proteins . However, M . pneumoniae possesses the HPr kinase/phosphorylase (HPrK/P), the key regulator of carbon metabolism in the Firmicutes . In contrast to the enzymes of other bacteria, the HPrK/P of M . pneumoniae is already active at very low ATP concentrations, suggesting a different mode of regulation . In this work, we studied the ability of M . pneumoniae to utilize different carbohydrates and their effects on the activity of the different phosphotransferase system (PTS) components . Glucose served as the best carbon source, with a generation time of about 30 h . Fructose and glycerol were also used but at lower rates and with lower yields . In contrast, M . pneumoniae is unable to use mannitol even though the bacterium is apparently equipped with all the genes required for mannitol catabolism . This observation is probably a reflection of the continuing and ongoing reduction of the M . pneumoniae genome . The general enzymatic and regulatory components of the PTS, i.e., enzyme I, HPr, and HPrK/P, were present under all growth conditions tested in this study . However, HPrK/P activity is strongly increased if the medium contains glycerol . Thus, the control of HPrK/P in vivo differs strongly between M . pneumoniae and the other Firmicutes . This difference may relate to the specific conditions on lipid-rich cell surfaces. J Appl Microbiol, 2004, 97(6), 1161 - 5 Development of a capture/enrichment sandwich ELISA for the rapid detection of enteropathogenic and enterohaemorrhagic Escherichia coli O26 strains; Neely E et al.; AIMS: To improve the sensitivity of a monoclonal antibody (MAb 2F3) based enteropathogenic Escherichia coli (EPEC)/enterohaemorrhagic E . coli (EHEC) serogroup O26-specific sandwich ELISA (sELISA), using a capture/enrichment format of the assay . METHODS AND RESULTS: The sELISA utilized an EPEC/EHEC O26-specific MAb 2F3 as the capture reagent and an E . coli serogroup O26 lipopolysaccharide-specific polyclonal antibody in the development stage . Wells containing faeces test samples from bovine enteritis cases and agar colony sweep cultures from human diarrhoea cases, after a 2-h capture stage, were washed and enrichment of the captured cells was encouraged by addition of tryptone soya broth . After overnight incubation, the contents of each well were transferred to sterile wells and the sELISA completed . Any sELISA positive samples were then subcultured onto blood agar to recover and further characterize the positive cultures . The assay had a sensitivity of 10(3) CFU ml(-1) . ELISA positive samples consisted of 21 (4.8%) of the 442 bovine and 19 (3.7%) of the 519 human samples tested, and ELISA positive EPEC/EHEC O26 strains were isolated from 11 and three of these samples respectively . CONCLUSION: The capture/enrichment method improved the sensitivity of a MAb-based sELISA for the detection of EPEC/EHEC O26 strains, and also contributed to an improved isolation rate of the organism from field samples . SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a specific MAb in a capture/enrichment format of the sELISA, provides a prospectively suitable screening method for the detection of pathogenic bacteria from mixed culture samples. Structure (Camb), 2004 Nov, 12(11), 1947 - 54 An alternate conformation and a third metal in PstP/Ppp, the M . tuberculosis PP2C-Family Ser/Thr protein phosphatase; Pullen KE et al.; Serine/threonine protein phosphatases are central mediators of phosphorylation-dependent signals in eukaryotes and a variety of pathogenic bacteria . Here, we report the crystal structure of the intracellular catalytic domain of Mycobacterium tuberculosis PstPpp, a membrane-anchored phosphatase in the PP2C family . Despite sharing the fold and two-metal center of human PP2Calpha, the PstPpp catalytic domain binds a third Mn(2+) in a site created by a large shift in a previously unrecognized flap subdomain adjacent to the active site . Mutations in this site selectively increased the Michaelis constant for Mn(2+) in the reaction of a noncognate, small-molecule substrate, p-nitrophenyl phosphate . The PstP/Ppp structure reveals core functional motifs that advance the framework for understanding the mechanisms of substrate recognition, catalysis, and regulation of PP2C phosphatases. Appl Environ Microbiol, 2004 Nov, 70(11), 6466 - 72 Measurements of fitness and competition in commensal Escherichia coli and E . coli O157:H7 strains; Durso LM et al.; Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied . It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts . To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains . The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E . coli and E . coli O157:H7 strains . Indirect antagonism assays revealed that E . coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity . Only minor differences were noted in the antibiotic resistance patterns of the two groups . In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles . Of 95 carbon sources tested, 27 were oxidized by commensal E . coli strains but not by the E . coli O157:H7 strains . Despite the observed physiological and biochemical differences between these two groups of E . coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment. Plant Physiol, 2004 Nov, 136(3), 3628 - 38 Epub 2004 Oct 29. Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense; Dong HP et al.; Ethylene (ET) signal transduction may regulate plant growth and defense, depending on which components are recruited into the pathway in response to different stimuli . We report here that the ET pathway controls both insect resistance (IR) and plant growth enhancement (PGE) in Arabidopsis (Arabidopsis thaliana) plants responding to harpin, a protein produced by a plant pathogenic bacterium . PGE may result from spraying plant tops with harpin or by soaking seeds in harpin solution; the latter especially enhances root growth . Plants treated similarly develop resistance to the green peach aphid (Myzus persicae) . The salicylic acid pathway, although activated by harpin, does not lead to PGE and IR . By contrast, PGE and IR are induced in both wild-type plants and genotypes that have defects in salicylic acid signaling . In response to harpin, levels of jasmonic acid (JA) decrease, and the COI1 gene, which is indispensable for JA signal transduction, is not expressed in wild-type plants . However, PGE and IR are stimulated in the JA-resistant mutant jar1-1 . In the wild type, PGE and IR develop coincidently with increases in ET levels and the expression of several genes essential for ET signaling . The ET receptor gene ETR1 is required because both phenotypes are arrested in the etr1-1 mutant . Consistently, inhibition of ET perception nullifies the induction of both PGE and IR . The signal transducer EIN2 is required for IR, and EIN5 is required for PGE because IR and PGE are impaired correspondingly in the ein2-1 and ein5-1 mutants . Therefore, harpin activates ET signaling while conscribing EIN2 and EIN5 to confer IR and PGE, respectively. J Food Prot, 2004 Oct, 67(10), 2274 - 6 Prevalence of Escherichia coli O157 in cattle and swine in central Mexico; Callaway TR et al.; Escherichia coli O157:H7 is a foodborne pathogenic bacterium that can reside undetected in the gastrointestinal tract of cattle because colonization by this bacterium is asymptomatic . Recent research has indicated that swine can carry and transmit this pathogen as well . The development of more advanced and sensitive detection techniques has improved the limit of detection and increased sensitivity for this important pathogen . This study was undertaken to determine the prevalence of E . coli O157 in cattle and swine in Mexico with the more sensitive detection technique of immunomagnetic bead separation . Samples (n = 60 per farm) were taken from four cattle and four swine farms (n = 240 cattle samples, n = 240 swine samples) located throughout central Mexico in October 2001 . The prevalence of E . coli O157 was found to be only 1.25% on cattle farms and 2.1% on swine farms . The prevalence in cattle in this study is lower than that reported in the United States and could be related to the lower reported prevalence of E . coli O157 in humans in Mexico . However, further research is needed to verify prevalence throughout other regions of Mexico, as well as prevalence during other seasons of the year. J Endod, 2004 Nov, 30(11), 770 - 3 Substance P enhances expression of lipopolysaccharide-induced inflammatory factors in dental pulp cells; Tokuda M et al.; To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures . Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA . We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity . In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs . In contrast, SP inhibited expression of interferon-gamma receptor mRNA . These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria. Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 1949 - 57 Epub 2004 Oct 20. Structure of 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase from Shewanella oneidensis at 1.6 A: identification of farnesyl pyrophosphate trapped in a hydrophobic cavity; Ni S et al.; Isopentenyl pyrophosphate (IPP) is a universal building block for the ubiquitous isoprenoids that are essential to all organisms . The enzymes of the non-mevalonate pathway for IPP synthesis, which is unique to many pathogenic bacteria, have recently been explored as targets for antibiotic development . Several crystal structures of 2C-methyl-D-erythritol-2,4-cyclophosphate (MECDP) synthase, the fifth of seven enzymes involved in the non-mevalonate pathway for synthesis of IPP, have been reported; however, the composition of metal ions in the active site and the presence of a hydrophobic cavity along the non-crystallographic threefold symmetry axis has varied between the reported structures . Here, the structure of MEDCP from Shewanella oneidensis MR1 (SO3437) was determined to 1.6 A resolution in the absence of substrate . The presence of a zinc ion in the active-site cleft, tetrahedrally coordinated by two histidine side chains, an aspartic acid side chain and an ambiguous fourth ligand, was confirmed by zinc anomalous diffraction . Based on analysis of anomalous diffraction data and typical metal-to-ligand bond lengths, it was concluded that an octahedral sodium ion was 3.94 A from the zinc ion . A hydrophobic cavity was observed along the threefold non-crystallographic symmetry axis, filled by a well defined non-protein electron density that could be modeled as farnesyl pyrophosphate (FPP), a downstream product of IPP, suggesting a possible feedback mechanism for enzyme regulation . The high-resolution data clarified the FPP-binding mode compared with previously reported structures . Multiple sequence alignment indicated that the residues critical to the formation of the hydrophobic cavity and for coordinating the pyrophosphate group of FPP are present in the majority of MEDCP synthase enzymes, supporting the idea of a specialized biological function related to FPP binding in a subfamily of MEDCP synthase homologs. Infect Immun, 2004 Nov, 72(11), 6262 - 70 Expression of type IV pili by Moraxella catarrhalis is essential for natural competence and is affected by iron limitation; Luke NR et al.; Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria . Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date . We identified and cloned the M . catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction . To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M . catarrhalis strain 7169 . Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M . catarrhalis expresses type IV pili that are essential for natural genetic transformation . Our studies suggest type IV pilus production by M . catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated . These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M . catarrhalis infections are warranted. Bioorg Med Chem, 2004 Nov 15, 12(22), 5865 - 73 Design, synthesis and biological evaluation of novel non-nucleoside HIV-1 reverse transcriptase inhibitors with broad-spectrum chemotherapeutic properties; Sriram D et al.; Acquired immunodeficiency syndrome (AIDS) results from infection by the retrovirus, human immunodeficiency virus (HIV) . HIV is the most significant risk factor for many opportunistic infections like tuberculosis, hepatitis, bacterial infections, etc . In this paper, we designed aminopyrimidinimino isatin lead compound as a novel non-nucleoside reverse transcriptase inhibitor with broad-spectrum chemotherapeutic properties for the effective treatment of AIDS and AIDS-related opportunistic infections . Compound 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7{{N(4)-{3'-(4'-amino-5'-trimethoxybenzylpyrimidin-2'-yl)imino-1'-(5-fluoroisatinyl)}methyl}-N(1)-piperazinyl}-3-quinoline carboxylic acid (12) emerged as the most potent broad-spectrum chemotherapeutic agent active against HIV, HCV, Mycobacterium tuberculosis and various pathogenic bacteria. Trends Microbiol, 2004 Nov, 12(11), 509 - 17 Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa; Ramos HC et al.; Flagella contribute to the virulence of pathogenic bacteria through chemotaxis, adhesion to and invasion of host surfaces . Flagellin is the structural protein that forms the major portion of flagellar filaments . Thus, flagellin consists of a conserved domain that is widespread in bacterial species and is dedicated to filament polymerization . Conversely, mammalian hosts detect the conserved domain on flagellin monomers through Toll-like receptor (TLR) 5, which triggers proinflammatory and adaptive immune responses . This review describes the relationships among flagellin molecular structure, bacterial virulence and host defenses, with special emphasis on mucosal tissues. Proc Natl Acad Sci U S A, 2004 Oct 19, 101(42), 15027 - 32 Epub 2004 Oct 11. A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles; Zhao X et al.; The rapid and sensitive determination of pathogenic bacteria is extremely important in biotechnology, medical diagnosis, and the current fight against bioterrorism . Current methods either lack ultrasensitivity or take a long time for analysis . Here, we report a bioconjugated nanoparticle-based bioassay for in situ pathogen quantification down to single bacterium within 20 min . The bioconjugated nanoparticle provides an extremely high fluorescent signal for bioanalysis and can be easily incorporated with biorecognition molecules, such as antibody . The antibody-conjugated nanoparticles can readily and specifically identify a variety of bacterium, such as Escherichia coli O157:H7, through antibody-antigen interaction and recognition . The single-bacterium-detection capability within 20 min has been confirmed by the plate-counting method and realized by using two independent optical techniques . The two detection methods correlated extremely well . Furthermore, we were able to detect multiple bacterial samples with high throughput by using a 384-well microplate format . To show the usefulness of this assay, we have accurately detected 1-400 E . coli O157 bacterial cells in spiked ground beef samples . Our results demonstrate the potential for a broad application of bioconjugated nanoparticles in practical biotechnological and medical applications in various biodetection systems . The ultimate power of integrating bionanotechnology into complex biological systems will emerge as a revolutionary tool for ultrasensitive detection of disease markers and infectious agents. J Anim Sci, 2004, 82 E-Suppl, E93 - 99 What are we doing about Escherichia coli O157:H7 in cattle? Callaway TR, Anderson RC, Edrington TS, Genovese KJ, Bischoff KM, Poole TL, Jung YS, Harvey RB, Nisbet DJ. Many human foodborne illnesses can be caused by consumption of foodstuffs (including meat products) contaminated with pathogenic bacteria from animal intestinal contents or hides . Steps that have been taken in the slaughter plant to decrease the spread of foodborne pathogenic bacteria (e.g., hazard analysis and critical control point methods) have been very effective; however, meat products are still the source of foodborne bacterial human illnesses . Increasing numbers of human Escherichia coli O157:H7 illnesses have also been related to contact with animals or to water supplies contaminated by run-off from cattle farms . Thus, strategies that specifically target foodborne pathogenic bacteria in the animal at the farm or feedlot level have great potential to improve food safety and decrease human illnesses . In this review, we describe a broad range of live-animal intervention strategies, both probiotic and antipathogen . Additionally, we examine some of the effects of diet and management strategies on foodborne pathogenic bacterial populations . The use of antibiotics in food animals to decrease foodborne pathogens also will be briefly examined . Overall, the concurrent use of several of these preslaughter intervention strategies could synergistically decrease human illnesses by providing for additional barriers in a multiple-hurdle approach to improving food safety. Trends Parasitol, 2004 Nov, 20(11), 531 - 6 Zoonotic protozoa: from land to sea; Fayer R et al.; Attention to worldwide pollution of the coastal marine environment has focused primarily on toxic algal blooms and pathogenic bacteria that multiply in nutrient-rich waters . However, massive but unseen amounts of feces from humans, their pets, and their domesticated animals are discharged, dumped, or carried in runoff, bringing encysted zoonotic protozoan parasites to estuaries and coastal waters . Here, they contaminate bathing beaches, are filtered and concentrated by shellfish eaten by humans and marine mammals, and infect a wide range of marine animal hosts, resulting in morbidity and mortality to some populations . This review addresses the extent of contamination and the animals affected by three genera of important zoonotic protozoa: Giardia, Cryptosporidium and Toxoplasma. Lasers Surg Med, 2004, 35(3), 206 - 13 Therapeutic ratio quantifies laser antisepsis: ablation of Porphyromonas gingivalis with dental lasers; Harris DM et al.; BACKGROUND AND OBJECTIVES: It is established that both pulsed Nd:YAG (1,064 nm) and continuous diode (810 nm) dental lasers kill pathogenic bacteria (laser antisepsis), but a quantitative method for determining clinical dosimetry does not exist . The purpose of this study was to develop a method to quantify the efficacy of ablation of Porphyromonas gingivalis (Pg) in vitro for two different lasers . STUDY DESIGN/MATERIALS AND METHODS: The ablation thresholds for the two lasers were compared in the following manner . The energy density was measured as a function of distance from the output of the fiber-optic delivery system . Pg cultures were grown on blood agar plates under standard anaerobic conditions . Blood agar provides an approximation of gingival tissue for the wavelengths tested in having hemoglobin as a primary absorber . Single pulses of laser energy were delivered to Pg colonies and the energy density was increased until the appearance of a small plume was observed coincident with a laser pulse . The energy density at this point defines the ablation threshold . Ablation thresholds to a single pulse were determined for both Pg and for blood agar alone . RESULTS: The large difference in ablation thresholds between the pigmented pathogen and the host matrix for pulsed-Nd:YAG represented a significant therapeutic ratio and Pg was ablated without visible effect on the blood agar . Near threshold the 810-nm diode laser destroyed both the pathogen and the gel . CONCLUSIONS: Clinically, the pulsed Nd:YAG may selectively destroy pigmented pathogens leaving the surrounding tissue intact . The 810-nm diode laser may not demonstrate this selectivity due to its greater absorption by hemoglobin and/or longer pulse duration . Nephrol Dial Transplant, 2004 Oct, 19(10), 2472 - 9 Reduced renal Na+-K+-Cl- co-transporter activity and inhibited NKCC2 mRNA expression by Leptospira shermani: from bed-side to bench; Wu MS et al.; BACKGROUND: Renal involvement is common in leptospirosis . Interstitial nephritis with interstitial oedema and mononuclear cellular infiltration are the usual findings . Clinically, non-oligouric acute renal failure, hypokalaemia and sodium wasting appear frequently in leptospirosis . The outer membrane protein from leptospira has been thought to be responsible for the disorder . However, the exact mechanisms of renal involvement are still unclear . METHODS: To address these questions, we first performed detailed in vivo clearance tests in three patients with leptospirosis (Leptospira shermani) and hypokalaemia to define the tubular lesion . These tests indicated a defective Na(+)-K(+)-Cl(-) co-transporter and a poor response to furosemide infusion . We performed further in vitro studies in a model of medullary thick ascending limb (mTAL) cultured cells derived from normal mouse . RESULTS: Outer membrane protein extract from L.shermani (0.3 microg/ml) inhibited Na(+)-K(+)-Cl(-) co-transporter activity in mTAL cells (control, 10.15+/-0.52; L.shermani, 6.47+/-0.47 nmol/min/mg protein) . The basolateral Na(+)-K(+) ATPase remained intact . Reverse transcription-polymerase chain reaction (RT-PCR) further revealed that the outer membrane protein extract from L.shermani downregulated Na(+)-K(+)-Cl(-) co-transporter (mNKCC2) mRNA expression . These changes were dose dependent and could be reversed by the antibody against outer membrane protein extract from L.shermani . Experiments with a less pathogenic strain of leptospira (L.bratislava) and Escherichia coli did not affect Na(+)-K(+)-Cl(-) co-transporter activity . CONCLUSIONS: We conclude that L.shermani leptospirosis downregulates mNKCC2 mRNA expression and inhibits Na(+)-K(+)-Cl(-) co-transporter activity in TAL cells . These alterations may explain the observed electrolyte disorders in leptospirosis. Blood . 2004 Sep 23; {Epub ahead of print} Impact of bone marrow hematopoiesis failure on T-cell generation during pathogenic simian immunodeficiency virus infection in macaques; Thiebot H et al.; Experimental infection of macaques with pathogenic strains of simian immunodeficiency virus (SIV) represents one of the most relevant animal models for study human immunodeficiency virus (HIV) pathogenesis . In this study, we demonstrated a significant decrease in the generation of CD4+ T cells from bone marrow CD34+ progenitors in macaques infected with SIVmac251 . This decrease appears to result from changes in the clonogenic potential of bone marrow progenitors of both the myeloid and lymphoid lineages . We also demonstrated a significant decrease in the numbers of the most immature long-term culture-initiating cells (LTC-IC) . Hematopoietic failure occurred as early as primary infection, in the absence of CD34+ bone marrow (BM) cell infection and was not related to plasma viral load . No major change was observed in the phenotype of BM CD34+ cells from infected macaques, although apoptosis markers appeared lowered, but a significantly higher that normal proportion of cells were in the G0/G1 . This is the first demonstration that bone marrow hematopoiesis failure results in impaired T-cell production, which may contribute to the disruption of T-lymphocyte homeostasis characteristic of pathogenic lentiviral infections in primates. Nat Rev Microbiol, 2004 Oct, 2(10), 833 - 41 Invertebrates as a source of emerging human pathogens; Waterfield NR et al.; Despite their importance, little is known about the origins of many emerging human pathogens . However, given the age and current predominance of invertebrates, it is likely that bacteria-invertebrate interactions are not only a present source of human pathogens but have also shaped their evolution . Pathogens of invertebrate and unicellular organisms represent an extensive reservoir of bacterial strains equipped with virulence factors that evolved to overcome the innate immune responses of their hosts . This reservoir might represent a source of new human pathogenic strains and might also foster the spread of novel virulence factors into existing human commensal or pathogenic bacteria . This article examines the available evidence for this concept by examining pairs of closely related bacteria, one of which is benign, but insect associated, and one of which is a human pathogen. Environ Res, 2004 Nov, 96(3), 250 - 6 Amoebae and other protozoa in material samples from moisture-damaged buildings; Yli-Pirila T et al.; Mold growth in buildings has been shown to be associated with adverse health effects . The fungal and bacterial growth on moistened building materials has been studied, but little attention has been paid to the other organisms spawning in the damaged materials . We examined moist building materials for protozoa, concentrating on amoebae . Material samples (n = 124) from moisture-damaged buildings were analyzed for amoebae, fungi, and bacteria . Amoebae were detected in 22% of the samples, and they were found to favor cooccurrence with bacteria and the fungi Acremonium spp., Aspergillus versicolor, Chaetomium spp., and Trichoderma spp . In addition, 11 seriously damaged samples were screened for other protozoa . Ciliates and flagellates were found in almost every sample analyzed . Amoebae are known to host pathogenic bacteria, such as chlamydiae, legionellae, and mycobacteria and they may have a role in the complex of exposure that contributes to the health effects associated with moisture damage in buildings. Front Biosci, 2004 Sep 01, 9, 2996 - 3006 Metal ion transport and regulation in mycobacterium tuberculosis; Agranoff D et al.; The regulation of metal ion concentrations is central to the physiology of the interaction between pathogenic bacteria and their hosts . Apart from the NRAMP orthologue, MntH, metal ion transporters in Mycobacterium tuberculosis have not been studied . Mn, the physiological substrate of MntH in other bacteria, may play an important role as a structural and redox-active cofactor in a wide range of metabolic processes . Fe, Cu and Zn play structural and catalytic roles in metalloenzymes involved in oxidative stress responses . Fe and Mg are required for growth in macrophages . Genomic analyses reveal 28 sequences encoding a broad repertoire of putative metal ion transporters (or transporter subunits), representing 24% of all transporters in this organism . These comprise 8 families of secondary active transporters and 3 families of primary active transporters, including 12,P, type ATPases . Potential metal ion specificities include K+, Na+, Cu2+, Cd2+, Zn2+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Fe2+/3+, Hg2+, AsO2- and AsO4(2-) . 17 of these transporters are also encoded as complete open reading frames in Mycobacterium leprae, suggesting a role in intracellular survival . Iron transcriptionally regulates a diverse set of genes via the iron-dependent DNA-binding proteins, Fur and IdeR . Changes in Fe and Mg concentrations signal entry into the intracellular compartment and potentially trigger up-regulation of virulence determinants . The plethora of putative transport systems encoded by the M . tuberculosis genome contrasts strikingly with the paucity of experimental data on these systems . The detailed analysis of the temporal pattern of M . tuberculosis transporter gene expression during infection will provide important insights into the basic biology of intracellular parasitism and may help to shape novel therapeutic strategies. Helicobacter, 2004, 9 Suppl 1, 7 - 14 Diagnosis of Helicobacter pylori Infection; Makristathis A et al.; While there are some attempts to improve culture of Helicobacter pylori, molecular methods have been the main focus of this interest . Their main application concerns the development of rapid tests also allowing the determination of bacterial resistance, i.e . real-time polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH), or to genotype the strains . Attempts to improve, simplify or explain the discrepancies of urea breath test results have been made and new generation of stool antigen test with monoclonal antibodies either using the standard ELISA format or rapid immunoenzymatic detection have confirmed their value . With regard to serology, studies have mainly focused on the distinction of infections with more pathogenic strains and the ability to diagnose atrophic gastritis with the Gastropanel. J Bacteriol, 2004 Sep, 186(18), 6118 - 23 Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12; Iqbal S et al.; It is known that Escherichia coli K-12 is cryptic (Phn-) for utilization of methyl phosphonate (MePn) and that Phn+ variants can be selected for growth on MePn as the sole P source . Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter . Here we show that Phn+ variants are present at the surprisingly high frequency of >10(-2) in K-12 strains . Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions . This revealed that, once selection for growth on MePn is removed, Phn+ revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence . It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization . The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate . All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences . The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn+ and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources. Clin Perinatol, 2004 Sep, 31(3), 489 - 500 Germ warfare: probiotics in defense of the premature gut; Hammerman C et al.; The potential benefits of a predominantly lactic acid bacterial flora include an improved balance of gut microbial ecology and decreased susceptibility of the gut mucosa to bacterial translocation via adherence to the intestinal mucosa, strengthening mucosal barrier function . These properties should be especially beneficial to the premature neonate with (1) delayed establishment of nor-mal flora, increasing the potential for proliferation of pathogenic bacteria and (2) immature development of the intestinal mucosa, rendering it more susceptible to the translocation of these pathogenic bacteria and leading to extra-intestinal spread and systemic disease . Early probiotic supplementation in preterm infants is theoretically sound and associated with minimal risk . Clinical data remain preliminary but are supportive of a reduction in feeding intolerance and NEC in this high-risk group. FEMS Immunol Med Microbiol, 2004 Sep 1, 42(1), 53 - 65 Ethnicity, infection and sudden infant death syndrome; Blackwell CC et al.; Epidemiological studies found the incidence of SIDS among Indigenous groups such as Aboriginal Australians, New Zealand Maoris and Native Americans were significantly higher than those for non-Indigenous groups within the same countries . Among other groups such as Asian families in Britain, the incidence of SIDS has been lower than among groups of European origin . Cultural and childrearing practices as well as socio-economic factors have been proposed to explain the greater risk of SIDS among Indigenous peoples; however, there are no definitive data to account for the differences observed . We addressed the differences among ethnic groups in relation to susceptibility to infection because there is evidence from studies of populations of European origin that infectious agents, particularly toxigenic bacteria might trigger the events leading to SIDS . The risk factors for SIDS parallel those for susceptibility to infections in infants, particularly respiratory tract infections which are also major health problems among Indigenous groups . Many of the risk factors identified in epidemiological studies of SIDS could affect three stages in the infectious process: (1) frequency or density of colonisation by the toxigenic species implicated in SIDS; (2) induction of temperature-sensitive toxins; (3) modulation of the inflammatory responses to infection or toxins . In this review we compare genetic, developmental and environmental risk factors for SIDS in ethnic groups with different incidences of SIDS: low (Asians in Britain); moderate (European/Caucasian); high (Aboriginal Australian) . Our findings indicate: (1) the major difference was high levels of exposure to cigarette smoke among infants in the high risk groups; (2) cigarette smoke significantly reduced the anti-inflammatory cytokine interleukin-10 responses which control pro-inflammatory responses implicated in SIDS; (3) the most significant effect of cigarette smoke on reduction of IL-10 responses was observed for donors with a single nucleotide polymorphism for the IL-10 gene that is predominant among both Asian and Aboriginal populations . If genetic makeup were a major factor for susceptibility to SIDS, the incidence of these deaths should be similar for both populations . They are, however, significantly different and most likely reflect differences in maternal smoking which could affect frequency and density of colonisation of infants by potentially pathogenic bacteria and induction and control of inflammatory responses. Acta Crystallogr D Biol Crystallogr, 1995 Nov, 51(Pt 6), 1097 - 8 X-ray analysis of crystals of polygalacturonase A from Pseudomonas solanacearum; Yoder MD; Crystals of the pectolytic protein, polygalacturonase A, have been obtained from polyethylene glycol 8000 using vapor diffusion methods . The 52.4 kDa protein is secreted by the plant pathogenic bacteria Pseudomonas solanacearum, and is important in the virulence of this plant pathogen . The protein crystallizes in space group P2(1) and has unit-cell parameters of a = 101.9, b = 124.6, c = 48.1 A, and beta= 105 degrees 50' . The crystal has two molecules in the asymmetric unit, and diffracts maximally to a resolution of 2.1 A. J Bacteriol, 2004 Aug, 186(16), 5442 - 9 DNA microarray-based genome comparison of a pathogenic and a nonpathogenic strain of Xylella fastidiosa delineates genes important for bacterial virulence; Koide T et al.; Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops . Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied . In this report, we show that X . fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants . Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12 . Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing . The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants . This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production . The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro . Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants . These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X . fastidiosa, constituting an important step towards understanding the molecular basis of the disease. J Bacteriol, 2004 Aug, 186(16), 5432 - 41 Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917; Grozdanov L et al.; Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases . To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E . coli strains by DNA-DNA hybridization . PCR-based screening of 324 nonpathogenic and pathogenic E . coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E . coli and also among extraintestinal and intestinal pathogenic strains . Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed . Comparison of these data with the available knowledge of the genome structure of E . coli K-12 strain MG1655 and of uropathogenic E . coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E . coli O6 strains . The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E . coli strain Nissle 1917. J Fish Dis, 2004 Aug, 27(8), 471 - 81 Humoral response and susceptibility of five full-sib families of Atlantic salmon, Salmo salar L., to the haemoflagellate, Cryptobia salmositica; Chin A et al.; Susceptibility and antibody production against pathogenic and vaccine strains of the haemoflagellate, Cryptobia salmositica were investigated in five full-sib families (A-E) of Atlantic salmon, Salmo salar . Humoral response and susceptibility of families were compared within three treatments: infection, vaccination and vaccination followed by challenge . Parasitaemias caused by the vaccine strain of C . salmositica were considerably lower than those caused by the pathogenic strain . All vaccinated families were protected when challenged with the pathogenic strain . Family B had significantly lower parasitaemias (with both strains) than the other families . When naive fish were infected with the pathogenic strain, this family had a significantly lower and earlier peak parasitaemia (4.3 +/-1.3 x 10(6) parasites mL(-1) blood at 3 weeks post-infection; w.p.i.) than the other families . Family C had the highest peak (11.1 +/- 1.2 x 10(6) parasites mL(-1) blood), which occurred at 4 w.p.i . Antibodies against C . salmositica were detected earlier in Family B (3 w.p.i.) than in Family C (5 w.p.i.) . This demonstrates an association of increased susceptibility with a delayed antibody response . Western immunoblot identified antibodies against 112, 181 and 200 kDa antigens earlier in more resistant fish (Family B) . Antigenic stimulation leading to a stronger antibody response was shown with the vaccine strain and in the later stages of infection. Annu Rev Phytopathol, 2004, 42, 339 - 66 Integrated approaches for detection of plant pathogenic bacteria and diagnosis of bacterial diseases; Alvarez AM; Disease diagnosis is based on a number of factors, including laboratory tests for pathogen identification . Rapid development of genomic techniques for characterization of bacteria over the past decade has greatly simplified and improved pathogen detection and identification, but DNA-based methods have not yet entirely replaced traditional culture and phenotypic tests in the plant industry . The first section of this review focuses on rapid immunodiagnostic and DNA-based detection methods for known bacterial pathogens in plants or plant products, which often manifest no symptoms of disease . The second section covers the broader topic of disease diagnosis and new methods for identifying and characterizing bacteria. EMBO J, 2004 Aug 4, 23(15), 2931 - 41 Epub 2004 Jul 22. A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin; Ahn VE et al.; The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl-chain recognition in any membrane-bound enzyme remains to be defined . PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin) . PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler . We present the 1.9 A crystal structure of PagP, an eight-stranded beta-barrel with an unexpected interior hydrophobic pocket that is occupied by a single detergent molecule . The buried detergent is oriented normal to the presumed plane of the membrane, whereas the PagP beta-barrel axis is tilted by approximately 25 degrees . Acyl group specificity is modulated by mutation of Gly88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition . A striking structural similarity between PagP and the lipocalins suggests an evolutionary link between these proteins. Anim Reprod Sci, 2004 Jul, 82-83, 295 - 306 Postpartum uterine health in cattle; Sheldon IM et al.; Uterine health is often compromised in cattle because postpartum contamination of the uterine lumen by bacteria is ubiquitous, and pathogenic bacteria frequently persist causing clinical disease . The subfertility associated with uterine infection involves perturbation of the hypothalamus, pituitary and ovary, in addition to the direct effects on the uterus, and appears to persist even after clinical resolution of the disease . Absorption of bacterial components from the uterus can prevent the follicular phase LH surge and ovulation . In addition, the first postpartum dominant follicle has a slower growth rate and secretes less estradiol at the end of the growth phase . There are also localised ovarian effects of high uterine bacterial growth density, because fewer first dominant follicles are selected in the ovary ipsilateral than contralateral to the previously gravid uterine horn . Thus, it is important to diagnose and treat uterine disease promptly and effectively . Examination of the contents of the vagina for the presence of pus is the most useful method for diagnosis of endometritis . The character and odor of the vaginal mucus can be scored and this endometritis score is correlated with the growth density of pathogenic bacteria in the uterus, and is prognostic for the likely success of treatment . The challenge for the future is to design prevention and control programs to reduce the incidence of disease, and understand how the immune and endocrine systems are integrated. J Virol, 2004 Aug, 78(15), 8372 - 81 Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus; Lee CW et al.; An outbreak of avian influenza (AI) caused by a low-pathogenic H5N2 type A influenza virus began in Mexico in 1993 and several highly pathogenic strains of the virus emerged in 1994-1995 . The highly pathogenic virus has not been reported since 1996, but the low-pathogenic virus remains endemic in Mexico and has spread to two adjacent countries, Guatemala and El Salvador . Measures implemented to control the outbreak and eradicate the virus in Mexico have included a widespread vaccination program in effect since 1995 . Because this is the first case of long-term use of AI vaccines in poultry, the Mexican lineage virus presented us with a unique opportunity to examine the evolution of type A influenza virus circulating in poultry populations where there was elevated herd immunity due to maternal and active immunity . We analyzed the coding sequence of the HA1 subunit and the NS gene of 52 Mexican lineage viruses that were isolated between 1993 and 2002 . Phylogenetic analysis indicated the presence of multiple sublineages of Mexican lineage isolates at the time vaccine was introduced . Further, most of the viruses isolated after the introduction of vaccine belonged to sublineages separate from the vaccine's sublineage . Serologic analysis using hemagglutination inhibition and virus neutralization tests showed major antigenic differences among isolates belonging to the different sublineages . Vaccine protection studies further confirmed the in vitro serologic results indicating that commercial vaccine was not able to prevent virus shedding when chickens were challenged with antigenically different isolates . These findings indicate that multilineage antigenic drift, which has not been observed in AI virus, is occurring in the Mexican lineage AI viruses and the persistence of the virus in the field is likely aided by its large antigenic difference from the vaccine strain. Spectrochim Acta A Mol Biomol Spectrosc, 2004 Jul, 60(8-9), 2153 - 62 Spectroscopic, redox and biological activities of transition metal complexes with ons donor macrocyclic ligand derived from semicarbazide and thiodiglycolic acid; Chandra S et al.; A novel macrocyclic Schiff base ligand (2,5,9,12,14,18-hexaoxo-7,16-dithia-1,3,4,10,11,13-hexaazacycloocta-decane (H6L) with N4S2 coordinating sites was prepared by the reaction of the semicarbazide and thiodiglycolic acid . The transition metal complexes with macrocyclic ligand were synthesized and characterized by elemental analyses, magnetic susceptibility measurements, molar conductance, IR, electronic, and EPR spectral studies . Mass, 1H NMR and IR spectral techniques suggest the structural features of macrocyclic ligand . Magnetic and electronic spectral studies suggest an octahedral geometry of complexes . Electrochemical behaviour of cobalt, nickel and copper complexes were determined by cyclic voltammetry . The cyclic voltammogram of the copper complex at room temperature shows a quasi-reversible peaks for Cu(III)-->Cu(II) and Cu(II)-->Cu(I) couples . The macrocyclic ligand and its complexes show growth inhibitory activity against pathogenic bacteria and plant pathogenic fungi A . niger, A . alternata and P . variotii . Most of the complexes have higher activities than that of free ligand. Indian J Exp Biol, 2003 Oct, 41(10), 1114 - 23 Molecular aspects of soybean cultivar-specific nodulation by Sinorhizobium fredii USDA257; Annapurna K et al.; Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules in association with the primitive soybean cultivar 'Peking' but fails to initiate nodules on many advanced soybean cultivars, including 'McCall' . This distinction is controlled by a set of nodulation genes termed nolXWBTUV . Inactivation of any of these genes enables USDA257 to nodulate McCall and many other improved soybean cultivars . Mutation in the nolXWBTUV locus also alters the Nod factor structure resulting in the production of a novel molecule with glucose incorporated into the chitin backbone . Some of the genes located in the nolXWBTUV locus reveal sequence homologies to known components of the type III secretion system (TTSS) of plant and animal pathogenic bacteria . Recent studies have demonstrated the presence of a complete TTSS in USDA257 and few other symbiotic bacteria . The TTSS cluster of USDA257 contains 27 open reading frames out of which 10 code for the structural components of the TTSS . USDA257, when grown in presence of flavonoids, secrete several proteins called Nops (Nodulation Outer Proteins) into the extracellular environment . Genes located in the TTSS of USDA257 encode some of the extracellular proteins, such as NopX, NopB, and NopL . These type III secreted proteins appear to play an important role in regulating nodulation in a host-dependent manner . Failure to elaborate the Nops results in a drastic phenotypic effect on soybean nodulation, indicating that these proteins may play a pivotal role in soybean cultivar specificity . The secretion of Nops appears to be facilitated by novel filamentous appendages (pili) that are produced by USDA257 upon induction by flavonoids . Biochemical studies have demonstrated the close association of several Nops with the purified pili . However, it remains to be seen if the filamentous appendages can function as conduits for delivery of Nops into the host cell . This review examines the current state of our knowledge on the molecular aspects of soybean cultivar-specific nodulation by USDA257. J Vet Med Sci, 2004 Jun, 66(6), 735 - 7 Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with proliferative enteropathy; Watarai M et al.; Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in domestic and experimental animals . Antiserum against synthetic peptides of the Lawsonia surface antigen (LsaA) well recognized L . intracellularis in infected ileum by immunohistochemistry . The synthetic peptides in LsaA showed strong reaction with serum from rabbits infected with L . intracellularis by enzyme-linked immunosorbent assay . These results suggest that ELISA used synthetic peptides in LsaA and anti-LsaA serum might be useful to diagnose for proliferative enteropathy. Vet Res, 2004 Jul-Aug, 35(4), 485 - 512 Equine Infectious Anemia Virus (EIAV): what has HIV's country cousin got to tell us? Leroux C, Cadore JL, Montelaro RC. Equine Infectious Anemia Virus (EIAV) is a lentivirus, of the Retrovirus family, with an almost worldwide distribution, infecting equids . It causes a persistent infection characterized by recurring febrile episodes associating viremia, fever, thrombocytopenia, and wasting symptoms . The disease is experimentally reproducible by inoculation of Shetland ponies or horses with EIAV pathogenic strains . Among lentiviruses, EIAV is unique in that, despite a rapid virus replication and antigenic variation, most animals progress from a chronic stage characterized by recurring peaks of viremia and fever to an asymptomatic stage of infection . The inapparent carriers remain infective for life, as demonstrated by experimental transfer of blood to naive animals . The understanding of the correlates of this immune control is of great interest in defining vaccine strategies . Research on EIAV, this "country cousin" of HIV (Human Immunodeficiency Virus), over the last five decades has produced some interesting results on natural immunological control of lentivirus replication and disease and on the nature and role of virus variation in persistence and pathogenesis . These studies are of interest in the context of HIV and efforts to develop a vaccine . This review will focus on some of the most recent results. Am J Physiol Cell Physiol, 2004 Nov, 287(5), C1404 - 11 Epub 2004 Jun 30. Human breast milk suppresses the transcriptional regulation of IL-1beta-induced NF-kappaB signaling in human intestinal cells; Minekawa R et al.; Neonatal necrotizing enterocolitis (NEC), which is a disease with a poor prognosis, is considered to be caused by the coincidence of intestinal ischemia-reperfusion injury and systemic inflammation due to the colonization of pathogenic bacteria . Interleukin (IL)-8, a proinflammatory cytokine, plays an important role in the pathophysiology of NEC . It was recently reported that IL-1beta activates the IL-8 gene by regulating the transcriptional nuclear factor kappaB (NF-kappaB) signaling pathways in intestinal cells . The protective role of maternal milk in NEC pathogenesis has been reported in both human and animal studies . In this study, we show that human breast milk dramatically suppressed the IL-1beta-induced activation of the IL-8 gene promoter by inhibiting the activation pathway of NF-kappaB . Moreover, we also show that human breast milk induced the production of IkappaBalpha . These results suggest that human breast milk could be protective and therapeutic in neonates with NEC by inhibiting the activation pathway of NF-kappaB. Mol Microbiol, 2004 Jul, 53(2), 623 - 38 Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori; Bury-Mone S et al.; The virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts . Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model . Little is known about how H . pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer . Thus, we compared gene expression profiles of H . pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real-time polymerase chain reaction, dot-blots or enzymatic assays . During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated . We found that acidity is a signal modulating the expression of several virulence factors . Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage . Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC-HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry . Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H . pylori . Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal-responsive transcriptional regulators (NikR and Fur) and an essential two-component response regulator (HP0166, OmpR-like) as effectors of the H . pylori acid response . Finally, a nikR-fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen. Eur J Nutr, 2004 Jun, 43 Suppl 2, II118 - II173 PASSCLAIM--gut health and immunity; Cummings JH et al.; BACKGROUND: The gut and immune system form a complex integrated structure that has evolved to provide effective digestion and defence against ingested toxins and pathogenic bacteria . However, great variation exists in what is considered normal healthy gut and immune function . Thus, whilst it is possible to measure many aspects of digestion and immunity, it is more difficult to interpret the benefits to individuals of variation within what is considered to be a normal range . Nevertheless, it is important to set standards for optimal function for use both by the consumer, industry and those concerned with the public health . The digestive tract is most frequently the object of functional and health claims and a large market already exists for gut-functional foods worldwide . AIM: To define normal function of the gut and immune system and describe available methods of measuring it . RESULTS: We have defined normal bowel habit and transit time, identified their role as risk factors for disease and how they may be measured . Similarly, we have tried to define what is a healthy gut flora in terms of the dominant genera and their metabolism and listed the many, varied and novel methods for determining these parameters . It has proved less easy to provide boundaries for what constitutes optimal or improved gastric emptying, gut motility, nutrient and water absorption and the function of organs such as the liver, gallbladder and pancreas . The many tests of these functions are described . We have discussed gastrointestinal well being . Sensations arising from the gut can be both pleasant and unpleasant . However, the characteristics of well being are ill defined and merge imperceptibly from acceptable to unacceptable, a state that is subjective . Nevertheless, we feel this is an important area for future work and method development . The immune system is even more difficult to make quantitative judgements about . When it is defective, then clinical problems ensure, but this is an uncommon state . The innate and adaptive immune systems work synergistically together and comprise many cellular and humoral factors . The adaptive system is extremely sophisticated and between the two arms of immunity there is great redundancy, which provides robust defences . New aspects of immune function are discovered regularly . It is not clear whether immune function can be "improved" . Measuring aspects of immune function is possible but there is no one test that will define either the status or functional capacity of the immune system . Human studies are often limited by the ability to sample only blood or secretions such as saliva but it should be remembered that only 2% of lymphocytes circulate at any given time, which limits interpretation of data.We recommend assessing the functional capacity of the immune system by: measuring specific cell functions ex vivo . measuring in vivo responses to challenge, e . g . change in antibody in blood or response to antigens . determining the incidence and severity of infection in target populations during naturally occurring episodes or in response to attenuated pathogens. Nat Prod Res, 2004 Aug, 18(4), 379 - 85 (22S)-6-O-acetyl-21betaH-hopane-3beta,6beta,22,29-tetrol from oakmoss (Evernia prunastri); Lutnaes BF et al.; A novel hopanoid triterpene, (22S)-6-O-acetyl-21betaH-hopane-3beta,6beta,22,29-tetraol, was isolated from oakmoss (Evernia prunastri (L.) Ach.), as identified from 1H, 13C, DEPT, COSY, NOESY, HSQC and HMBC NMR, MS and IR spectroscopy . During recrystallisation a new compound, 30-nor-6-O-acetyl-3beta,6beta-dihydroxy-21alphaH-hopan-22-one, was formed by a formal loss of methanol from the dihydroxypropyl moiety . No biological activity was found for the naturally occurring compound upon testing against a series of fish and human pathogenic bacteria. Bacteriol Virusol Parazitol Epidemiol, 2003 Jan-Mar, 48(1), 31 - 6 {Uropathogenic Escherichia coli--ExPEC pathotype, the best characterized}; Usein CR et al.; Uropathogenic E . coli strains are included in the recently defined pathotype ExPEC together with other E . coli isolates involved in various extraintestinal infections . These strains possess specific virulence factors such as adherence systems, toxins, iron uptake systems that distinguish them from the commensal strains . The expression of bacterial adherence structures requires a complex organization which involves a different number of proteins depending upon the assembly in fimbrial protrudings or afimbrial aggregates . However one could notice that their genetic determinants are usually similar . The toxins synthetized by ExPEC strains, mainly represented by hemolysin and cytotoxic necrotizing factor contribute to destruction of eukaryotic cells . Such siderophores as aerobactin confer the strains the advantage of capturing iron from the environment in which they must survive and replicate . Co-expression of virulence factors contributes to the host's defence system defeat and onset of infection . Quite often the codifying genes are localized in distinctive regions on the bacterial chromosome called pathogenicity-associated islands . The horizontal transfer of these DNA blocks among individuals belonging to the same species is one of the processes involved in the evolution of pathogenic strains. AIDS, 2004 Jul 2, 18(10), 1363 - 70 HIV in central nervous system and behavioral development: an HIV-2287 macaque model of AIDS; Kinman LM et al.; OBJECTIVE: To determine which route of inoculation produced consistent and frequent HIV infection in the central nervous system (CNS) and alterations in cognitive and motor development in infant macaques . METHODS: Infant macaques (Macaca nemestrina) were inoculated with the highly pathogenic strain HIV-2287 intravenously (n = 3) or intrathecally (n = 3) . Uninfected infants were evaluated as controls . Disease progression was evaluated by virological assessment of blood and cerebral spinal fluid (CSF), CD4 T cell count in blood, and quinolinic acid levels in CSF (a surrogate marker of neuronal cell damage) . The effect of HIV infection on cognitive and motor development in infants was monitored during the 6-month study . RESULTS: Either route of HIV-2287 inoculation produced detectable viral RNA in CSF and productive infection in blood . Detection of virus in CSF paralleled a rise in quinolinic acid levels . All HIV-infected infants experienced a severe and rapid decline in CD4 T cell counts by 10 weeks after viral infection . HIV-infected infants, particularly those infected by the intravenous route, exhibited delays in reaching cognitive and motor milestones, which paralleled neuropathological changes . CONCLUSIONS: The HIV-2287 infant model produced a high incidence of viral infection in the CNS regardless of the route of inoculation . Significant alteration in neurobehavioral development was observed in HIV-infected infants, and this measure was significantly impaired particularly in infants infected by the intravenous route . These data, coupled with the ability to detect viral RNA and changes in quinolinic acid levels in CSF, may allow quantitative evaluation of drug and immune candidates for treating neurological effects of AIDS. Chin Med J (Engl), 2004 Jun, 117(6), 828 - 34 Protective effect of a bacterial extract against acute exacerbation in patients with chronic bronchitis accompanied by chronic obstructive pulmonary disease; Li J et al.; BACKGROUND: Immunostimulating agents made from bacterial extracts represent a class of medications that contains antigens derived from several bacterial strains and their potential ability to prevent bacterial infections results from the stimulation of the nonspecific component of the immune system . The present study investigated the effect of the oral immunostimulant Broncho-Vaxom, which includes material from eight different species of bacteria that are frequently present in the lower respiratory tract, on the frequency and severity of acute exacerbation in patients with chronic bronchitis accompanied by chronic obstructive pulmonary disease (COPD) . METHODS: Ninety patients with chronic bronchitis complicated with COPD were randomly divided into groups A and B . Forty-nine subjects in group A received oral capsules containing 7 mg Broncho-Vaxom, while 41 patients in group B received similar placebo capsules . Both groups took one capsule daily for the first 10 days of each month for 3 consecutive months . The frequency of acute exacerbation, symptom scores, and lung function were recorded for the following one year period . RESULTS: There was a significant decrease in the incidence, duration, and severity of acute exacerbation, as well as a reduction in the course of antibiotics administered and in the dosage of bronchodilator and mucolytic agent in group A, as compared to group B (P < 0.05, respectively) . Symptom scores for cough, sputum, dyspnea, as well as symptoms observed upon auscultation of the chest also improved significantly in group A as compared to group B (P < 0.05, respectively) . The bacterial clearance rate in sputum cultures from patients who received no antibiotics for the first 3 months was also significantly higher in group A compared to group B (P < 0.01) . CONCLUSIONS: Orally administered Broncho-Vaxom is associated with a decrease in the incidence of acute exacerbation and a decrease in the need for antibiotics and symptomatic relief medications in patients with chronic bronchitis accompanied by COPD . Broncho-Vaxom is also associated with a decrease in symptom scores . Without causing any apparent adverse effects, this drug may also help to eradicate pathogenic bacteria in the airways. Curr Opin Microbiol, 2004 Jun, 7(3), 290 - 5 Laser microdissection: exploring host-bacterial encounters at the front lines; Hooper LV; The mucosal surfaces of tissues such as the stomach and intestines are in constant contact with indigenous bacterial populations and are major portals of entry for bacterial pathogens . Host responses to bacterial encounters at these surfaces frequently involve complex interactions between epithelial cells and immune cells, and are thus difficult to model in vitro . Laser microdissection is a technique in which pure populations of host cells are acquired from sections of complex tissue . When coupled with an expanding repertoire of techniques for molecular analysis of microdissected cells, laser microdissection allows host cellular responses to bacteria to be studied in their native tissue context . This approach has already yielded key insights into the nature of mucosal responses to commensal, as well as pathogenic bacteria, and promises to be an important addition to the cellular microbiologist's toolkit. Microbiology, 2004 Jun, 150(Pt 6), 1707 - 12 Naturally occurring horizontal gene transfer and homologous recombination in Mycobacterium; Krzywinska E et al.; Acquisition of genetic information through horizontal gene transfer (HGT) is an important evolutionary process by which micro-organisms gain novel phenotypic characteristics . In pathogenic bacteria, for example, it facilitates maintenance and enhancement of virulence and spread of drug resistance . In the genus Mycobacterium, to which several primary human pathogens belong, HGT has not been clearly demonstrated . The few existing reports suggesting this process are based on circumstantial evidence of similarity of sequences found in distantly related species . Here, direct evidence of HGT between strains of Mycobacterium avium representing two different serotypes is presented . Conflicting evolutionary histories of genes encoding elements of the glycopeptidolipid (GPL) biosynthesis pathway led to an analysis of the GPL cluster genomic sequences from four Mycobacterium avium strains . The sequence of M . avium strain 2151 appeared to be a mosaic consisting of three regions having alternating identities to either M . avium strains 724 or 104 . Maximum-likelihood estimation of two breakpoints allowed a approximately 4100 bp region horizontally transferred into the strain 2151 genome to be pinpointed with confidence . The maintenance of sequence continuity at both breakpoints and the lack of insertional elements at these sites strongly suggest that the integration of foreign DNA occurred by homologous recombination . To our knowledge, this is the first report to demonstrate naturally occurring homologous recombination in Mycobacterium . This previously undiscovered mechanism of genetic exchange may have major implications for the understanding of Mycobacterium pathogenesis. J Hosp Infect, 2004 Jun, 57(2), 162 - 9 Misuse of gloves: the foundation for poor compliance with hand hygiene and potential for microbial transmission? Girou E, Chai SH, Oppein F, Legrand P, Ducellier D, Cizeau F, Brun-Buisson C. Improvement in hand hygiene compliance is important for reducing cross-infection by micro-organisms . The objective of this prospective observational study was to measure how the improper use of gloves limits compliance to hand hygiene and exposes patient's to infection . The study was conducted in five wards (three intensive care units and two medical wards) in a French university hospital . Staff-patient and staff-environment contacts were observed in 120 healthcare workers caring for patients colonized or infected with pathogenic bacteria . Hand hygiene was not undertaken due to improper gloving in 64.4% (95%CI, 64.1% to 65.1%) of instances . Possible microbial transmission might have occurred in 18.3% (95%CI, 17.8% to 18.8%) of all contacts because used gloves were not removed before performing care activities that necessitated strict aseptic precautions . Failure to change or remove contaminated gloves was a major component in the poor compliance with hand hygiene and carried a high-risk of microbial transmission . Improving hand hygiene compliance will require changing healthcare workers behaviour towards glove use . Science, 2004 Jun 4, 304(5676), 1515 - 8 A process for controlling intracellular bacterial infections induced by membrane injury; Roy D et al.; Strategies for inhibiting phagolysosome fusion are essential for the intracellular survival and replication of many pathogens . We found that the lysosomal synaptotagmin Syt VII is required for a mechanism that promotes phagolysosomal fusion and limits the intracellular growth of pathogenic bacteria . Syt VII was required for a form of Ca2+-dependent phagolysosome fusion that is analogous to Ca2+-regulated exocytosis of lysosomes, which can be triggered by membrane injury . Bacterial type III secretion systems, which permeabilize membranes and cause Ca2+ influx in mammalian cells, promote lysosomal exocytosis and inhibit intracellular survival in Syt VII +/+ but not -/- cells . Thus, the lysosomal repair response can also protect cells against pathogens that trigger membrane permeabilization. FEBS Lett, 2004 Jun 4, 567(2-3), 297 - 301 BOF: a novel family of bacterial OB-fold proteins; Ginalski K et al.; Using top-of-the-line fold recognition methods, we assigned an oligonucleotide/oligosaccharide-binding (OB)-fold structure to a family of previously uncharacterized hypothetical proteins from several bacterial genomes . This novel family of bacterial OB-fold (BOF) proteins present in a number of pathogenic strains encompasses sequences of unknown function from DUF388 (in Pfam database) and COG3111 . The BOF proteins can be linked evolutionarily to other members of the OB-fold nucleic acid-binding superfamily (anticodon-binding and single strand DNA-binding domains), although they probably lack nucleic acid-binding properties as implied by the analysis of the potential binding site . The presence of conserved N-terminal predicted signal peptide indicates that BOF family members localize in the periplasm where they may function to bind proteins, small molecules, or other typical OB-fold ligands . As hypothesized for the distantly related OB-fold containing bacterial enterotoxins, the loss of nucleotide-binding function and the rapid evolution of the BOF ligand-binding site may be associated with the presence of BOF proteins in mobile genetic elements and their potential role in bacterial pathogenicity. Mar Environ Res, 2004 Aug-Dec, 58(2-5), 719 - 23 Mycobacteria, but not mercury, induces metallothionein (MT) protein in striped bass, Morone saxitilis, phagocytes, while both stimuli induce MT in channel catfish, Ictalurus punctatus, phagocytes; Regala RP et al.; Recent advances in molecular immunology indicate that the expression of inducible pro-inflammatory proteins is increased in vertebrates in response to both infectious disease agents and various xenobiotics . For example, iNOS, COX-2, and CYP1A are induced by both inflammation and AhR ligands . Moreover, the expression of these proteins in response to stimuli varies among individuals within populations . Little is known of the differences among fish in the inducibility of proinflammatory proteins in response to both infectious agents and xenobiotics . Through random screening of a striped bass, Morone saxitilis, peritoneal macrophage cDNA library, a full length metallothionein (MT) gene was cloned and sequenced . MT is a low-molecular weight (6-8 kDa), cysteine-rich metal binding protein . Metals are required by pathogenic bacteria for growth, and by the host defense system by serving as a catalyst for the generation of reactive oxygen intermediates (ROIs) by phagocytes . A recombinant striped bass MT (rMT) was expressed and purified, then used to generate a specific mAb (MT-16) . MT protein expression was followed in freshly isolated striped bass and channel catfish, Ictalurus punctatus, phagocytes after in vitro exposure to the naturally occurring intracellular pathogen Mycobacteria fortuitum or to 0.1 and 1 microM mercury (Hg), as HgCl(2) . MT expression was increased by 24 h in both channel catfish and striped bass phagocytes as a result of exposure to M . fortuitum cells . On the other hand, MT was induced by Hg in channel catfish cells, but not those of striped bass . These results indicate that metal homeostasis in phagocytes is different between catfish and striped bass . In addition, these data suggest that care should be taken to distinguish between inflammation-induced vs . metal-induced MT when using MT expression as a biomarker of metal exposure. J Mol Evol, 2004 May, 58(5), 596 - 605 Ectopic gene conversions in four Escherichia coli genomes: increased recombination in pathogenic strains; Morris RT et al.; We characterized the ectopic gene conversions in the genomes of the K-12 MG1655, O157:H7 Sakai, O157:H7 EDL933, and CFT073 strains of E coli . Compared to the three pathogenic strains, the K-12 strain has a much smaller number of gene families, its gene families contain fewer genes, and gene conversions are less frequent . Whereas the three pathogenic strains have gene conversions covering hundreds of nucleotides when their flanking regions have as little as 50% similarity, flanking region similarity of at least 94% on both sides of the converted region is required to observe conversions of more than 87 nucleotides in the K-12 strain . Recombination is therefore more frequent and requires less sequence similarity in the three pathogenic strains than in K-12 . This higher recombination level might be due to mutations in some of their mismatch-repair genes . In contrast with the gene conversions present in the yeast genome, the gene conversions found in the E . coli genomes do not occur more frequently between duplicated genes that are close to one another than between duplicated genes that are far apart and are randomly distributed along the length of the genes . In E . coli, gene conversions are not more frequent near the origin of replication . However, they do occur more frequently near the terminus of replication of the Sakai genome, where multigene family members are more abundant . This suggests that, in E . coli, gene conversions occur randomly between genes located in different chromosomal locations or located on different copies of the multiple chromosomes found in E . coli cells. Cell, 2004 May 28, 117(5), 558 - 9 Class II MHC: sweetening the peptide only diet? Watts C. MHC molecules typically bind peptides to create ligands for the T cell antigen receptor . In this issue of Cell, report an unexpected association of class II MHC molecules with processed zwitterionic polysaccharides from pathogenic bacteria . The complexes appear to modulate the T cell dependent pathology of abscess formation. Diabetes Metab Res Rev, 2004 May-Jun, 20 Suppl 1, S78 - 89 Wound healing and treatments for people with diabetic foot ulcers; Jeffcoate WJ et al.; The factors that delay wound healing are multiple and relate both to diabetes and to the effect of its complications . Diabetic foot ulcers readily become chronic, and chronic ulcers have biological properties that differ substantially from acute ones . Much of the available information on the biology of wound healing relates to acute and experimental wounds and may not be directly relevant . It follows that there is limited evidence currently available to underpin protocols for the management of diabetic foot ulcers, or to guide choice of applications and dressings 1 . Nevertheless, it is possible to define certain principles.GLYCAEMIC CONTROL: The first relates to glycaemic control . While chronic complications of diabetes such as peripheral vascular disease and neuropathy may be largely irreversible, aspects of structure and function of connective tissue and cells may be impaired by hyperglycaemia, and their function should be improved if normoglycaemia is achieved . PROMOTION OF HEALING: The second principle concerns attempts at active promotion of wound healing by (1) surgical revascularization, and (2) specific attempts to correct defined biological abnormalities thought to be hindering the healing process . These include the use of a variety of applications, dressings and technologies, which may stimulate healing by applying, or stimulating the release of, growth factors and cytokines . While this approach holds the greatest promise for the future, it will be dependent on defining defects which need correction in specific individuals, and having technologies available to address them . This field is in its infancy . WOUND CARE: The third broad principle concerns the management of the wound and its surrounding tissue in order to promote healing . This includes regular inspection, cleansing and removal of surface debris, elimination of pathogenic bacteria and creation of an appropriate environment to facilitate endogenous tissue regeneration . There are many applications and dressings that may be chosen to promote healing, but, whichever is selected, wound management has to be integrated into an effective programme of multidisciplinary care . J Bacteriol, 2004 Jun, 186(11), 3590 - 8 Methionine sulfoxide reductase A (MsrA) deficiency affects the survival of Mycobacterium smegmatis within macrophages; Douglas T et al.; Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme which reduces oxidized methionine to methionine . Since oxidation of methionine in proteins impairs their function, an absence of MsrA leads to abnormalities in different organisms, including alterations in the adherence patterns and in vivo survival of certain pathogenic bacteria . To understand the role of MsrA in intracellular survival of bacteria, we disrupted the gene encoding MsrA in Mycobacterium smegmatis through homologous recombination . The msrA mutant strain of M . smegmatis exhibited significantly reduced intracellular survival in murine J774A.1 macrophages compared to the survival of its wild-type counterpart . Furthermore, immunofluorescence and immunoblotting of phagosomes containing M . smegmatis strains revealed that the phagosomes with the msrA mutant strain acquired both p67(phox) of phagocyte NADPH oxidase and inducible nitric oxide synthase much earlier than the phagosomes with the wild-type strain . In addition, the msrA mutant strain of M . smegmatis was observed to be more sensitive to hydroperoxides than the wild-type strain was in vitro . These results suggest that MsrA plays an important role in both extracellular and intracellular survival of M . smegmatis. J Bacteriol, 2004 Jun, 186(11), 3472 - 9 Characterization of a eukaryotic-like tyrosine protein kinase expressed by the Shiga toxin-encoding bacteriophage 933W; Tyler JS et al.; The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs) . Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer . We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase . An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues . A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs . We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny . The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria. Int J Med Microbiol, 2004 Apr, 293 Suppl 37, 109 - 16 Lyme borreliosis spirochetes in questing ticks from mainland Portugal; Baptista S et al.; In Portugal, Ixodes ricinus ticks have been shown to contain DNA of several spirochetes belonging to the Borrelia burgdorferi sensu lato complex, with major differences in the genetic diversity between ecozones . Some isolates have been obtained since 1999, confirming the circulation of pathogenic strains in these ticks . Ixodes ricinus is considered to be a widespread species, however, in Portugal it is found only in a few habitats . Here we present preliminary results from a nationwide survey of questing I . ricinus (n = 4,001) and other Ixodidae (n = 1,534) in Portugal, initiated in 2001 . The sampling points (so far 41) were selected using a Geographic Information System, according to the type of vegetation, accessibility and prevalence of human cases . The spatial and temporal of tick abundance and the infection of B . burgdorferi sensu lato in ticks were determined in selected areas . Ticks were examined for the presence of B . burgdorferi sensu lato by culturing (719 out of 4,001 I . ricinus), and direct PCR amplification of the 5S-23S intergenic spacer region (1,870 out of 5,535) followed by RFLP analysis, the reverse line blot assay and nucleotide sequencing of PCR amplicons . The most abundant tick genus was Rhipicephalus (53%), followed by Dermacentor (34%), I . ricinus and Hyalomma (7%, each) . The Mafra and Grandola sites, where a more intensive study was carried out, were excellent habitats for I . ricinus . However, a clear difference of the prevalence of Borrelia infection and the genetic diversity of circulating spirochetes was observed in these two sites . Genotyping of all I . ricinus isolates revealed 5 B . garinii, 8 B . lusitaniae and 1 B . valaisiana strains, which were obtained for the first time in these regions along with a considerable percentage of tick-derived PCR amplicons . Two hard-tick species other than I . ricinus in Grandola were also B . lusitaniae positive, thus seeming to take part in the transmission cycle of Borrelia . The seasonal dynamics of I . ricinus in Mafra was bimodal, more pronounced in nymphs than in adults . The present findings indicate that B . burgdorferi sensu lato agents are differentially maintained in nature in local tick populations in different geographic areas across Portugal and that the risk of acquiring Lyme borreliosis in certain areas of Portugal is higher than previously assumed. Clin Diagn Lab Immunol, 2004 May, 11(3), 532 - 7 2F3 monoclonal antibody recognizes the O26 O-antigen moiety of the lipopolysaccharide of enterohemorrhagic Escherichia coli strain 4276; Szalo IM et al.; Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E . coli (EPEC) organisms are groups of pathogenic strains whose infections are characterized by a typical lesion of enterocyte attachment and effacement . They are involved in enteric diseases both in humans and in animals, and EHEC strains can be responsible for hemolytic uremic syndrome in humans . Previously, it was shown that the 2F3 monoclonal antibody (MAb) is specific for the O26 EHEC and EPEC strains (P . Kerr, H . Ball, B . China, J . Mainil, D . Finlay, D . Pollock, I . Wilson, and D . Mackie, Clin . Diagn . Lab . Immunol . 6:610-614, 1999) . As these groups of bacteria play an important role in pathology, the aim of this paper was to characterize the antigen recognized by the 2F3 MAb and its genetic determinant . A genomic locus containing the entire O-antigen gene cluster and half of the colanic acid gene cluster from an O26 EHEC strain was shown to be sufficient for the production of the antigen recognized by the 2F3 MAb in an E . coli DH5alpha strain . By transposon mutagenesis performed on the recombinant plasmid, all 2F3 enzyme-linked immunosorbent assay-negative mutants had their transposons inserted into the O-antigen gene cluster . The O-antigen gene cluster was also cloned from an O26 EHEC strain into the E . coli DH5alpha strain, which then produced a positive result with the 2F3 MAb . Further analysis of the type of lipopolysaccharides (smooth or rough) produced by the clones and mutants and of the O antigen of the 2F3-positive clones confirmed that the epitope recognized by the 2F3 MAb is located on the O antigen in the O26 EHEC and EPEC strains and that its genetic determinant is located inside the O-antigen gene cluster. J Mol Biol, 2004 May 28, 339(2), 459 - 69 Sensor domain of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknD, forms a highly symmetric beta propeller; Good MC et al.; Diverse pathogenic bacteria produce transmembrane receptor Ser/Thr protein kinases (STPKs), but little is known about the signals mediated by these "eukaryotic-like" proteins . To explore the basis for signaling in the bacterial STPK receptor family, we determined the structure of the sensor domain of Mycobacterium tuberculosis PknD . In two crystal forms, the PknD sensor domain forms a rigid, six-bladed beta-propeller with a flexible tether to the transmembrane domain . The PknD sensor domain is the most symmetric beta-propeller structure described . All residues that vary most among the blade subdomains cluster in the large "cup" motif, analogous to the ligand-binding surface in many beta-propeller proteins . These results suggest that PknD binds a multivalent ligand that signals by changing the quaternary structure of the intracellular kinase domain. Int J Food Microbiol, 2004 May 15, 93(1), 73 - 85 Modelling effect of physical and chemical parameters on heat inactivation kinetics of hepatitis A virus in a fruit model system; Deboosere N et al.; While thermal destruction of pathogenic bacteria has been thoroughly studied in food industry, heat inactivation of viruses in food has been poorly investigated . Experiments were carried out to characterize the effects of controlled physical and chemical characteristics of a food matrix upon heat resistance parameters (D and z values) of hepatitis A virus (HAV), taken as model because of its reported heat resistance . Sucrose content (28-52 degrees Brix), calcium concentration (90-1700 mg kg(-1)) and pH (3.3-4.3) were selected for possible influence on thermal inactivation of HAV in strawberry mashes and thus included in an experimental design according to a Doehlert matrix . Use of this design not only allowed to detect and quantify the direct influence of sucrose concentration upon the D85 degrees C value to be higher than the one of pH, but also to reveal a sucrose concentration/pH specific interaction, while no effect of calcium concentration was evidenced . Although the model cannot be directly used to predict heat resistance in real fruit systems, because of differences observed between predicted and measured D85 degrees C values, it is useful for predicting the trends and relative changes in D values due to sucrose concentration and pH variations . Results suggested possible effects of other constituents of strawberry products on heat resistance of HAV and confirmed the importance of experimental validation of any model-derived process . Nevertheless, such a modelling approach using response surface methodology provides a rapid answer to heat resistance evaluation of a food-borne virus as a function of specific physical and chemical parameters of specific food products . Tsitol Genet, 2004 Mar-Apr, 38(2), 65 - 80 {Modification of lipopolysaccharides as one of the ways of adaptation of pathogenic bacteria and their populations to biotic and abiotic environmental factors}; Shylina IuV et al.; The review is devoted to a problem of adaptive role of lipopolysaccharide (LPS) modification of pathogenic bacteria cells . The biological activity of LPS depends on their molecular conformation, which is determined by primary structure and charges of their molecules and can change through regulation of enzyme activity of LPS separate component synthesis . Regulation of gene expression, which determines LPS synthesis, can occur at a level of DNA primary structure (through mutations and recombinations); on highest levels of DNA organization through alteration of its conformation; can be mediated by signal transduction systems of bacterial cells . The changes of LPS structure can have spontaneous character due both to the instability of the appropriate genes and to the induction under the influence of various factors . Spontaneous variation of LPS structure is one of the reasons of heterogeneity of bacterial cell populations and creates a basis of their preadaptation . The influence of the environment factors on structure and functions of LPS can be both direct and mediated through regulation of expression of chromosome and plasmid genes by direct influence of the factors on DNA and through signal transduction systems of bacterial cells . Pathogenic bacteria can use the LPS variations at adaptation to biotic and abiotic factors. Int J Pediatr Otorhinolaryngol, 2004 Mar, 68(3), 331 - 9 Otitis media with effusion: disease or defense? A review of the literature; de Ru JA et al.; Many studies of otitis media with effusion (OME) have been published, most of them dealing with risk factors . The literature correlates this condition with various patient characteristics and socio-economic factors, but none of these have been identified as the sole causative factor . A review of the literature suggests that otitis media with effusion is a response to pathogenic bacteria and thus a normal protective reaction of the body . Therefore, the decision on whether or not treatment is indicated should take the natural course of that response into account . In light of the literature reviewed here, we conclude that there is usually no need to treat middle ear effusion in young children. Proc Natl Acad Sci U S A, 2004 May 11, 101(19), 7252 - 7 Epub 2004 Apr 27. Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers; Leduc D et al.; Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5'-monophosphate) synthesis in a large number of microbial species . Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis . By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers . Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth. J Mol Biol, 2004 May 21, 339(1), 207 - 15 Unique structural characteristics of peptide deformylase from pathogenic bacterium Leptospira interrogans; Zhou Z et al.; Peptide deformylase (PDF), which is essential for normal growth of bacteria but not for higher organisms, is explored as an attractive target for developing novel antibiotics . Here, we present the crystal structure of Leptospira interrogans PDF (LiPDF) at 2.2A resolution . To our knowledge, this is the first crystal structure of PDF associating in a stable dimer . The key loop (named the CD-loop: amino acid residues 66-76) near the active-site pocket adopts "closed" or "open" conformations in the two monomers forming the dimer . In the closed subunit, the CD-loop and residue Arg109 block the entry of the substrate-binding pocket, while the active-site pocket of the open subunit is occupied by the C-terminal tail from the neighbouring molecule . Moreover, a formate group, as one product of deformylisation, is observed bound with the active-site zinc ion . LiPDF displays significant structural differences in the C-terminal region compared to both type-I and type-II PDFs, suggesting a new family of PDFs. Best Pract Res Clin Gastroenterol, 2004 Apr, 18(2), 373 - 86 Microbial-gut interactions in health and disease . Epithelial cell responses; Mavris M et al.; Pathogenic bacteria use many strategies to secure their survival within the host . Enteropathogens exploit intestinal epithelial cells in many ways, including the manipulation of normal cellular functioning, or of cellular structural components, or by the induction of signalling pathways, such as the production of pro-inflammatory cytokines . However, the enterocyte warns the host of impending danger and, in turn, elicits a protective response . Pathogens are detected by epithelial cells owing to their vast array of surface antigens and secreted products . Epithelial cells have developed both extracellular and intracellular sensing proteins that function as a first line of defence against pathogens; this is followed by acquired immunity, namely IgA, which is used as reinforcement . Thus, in a game of constant attack and defence, the pathogen and the enterocyte aim to outsmart each other in an effort to survive. Med Oral, 2004 May-Jul, 9(3), 191 - 6 Optimal assay conditions for quantifying fibronectin in saliva; Llena-Puy MC et al.; Introduction: Fibronectin (Fn) is a glycoprotein that is present in many body fluids and tissues in both physiological and pathological conditions . It can also be detected in the saliva, although only in very small quantities and frequently in broken chains . It induces bacterial aggregation and its levels fall when those of cariogenic or periodontal pathogenic bacteria rise . The infective capacity of the saliva of patients infected by human immunodeficiency virus (HIV) has been linked to the levels of this protein . In some chronic conditions of the oral mucosa,such as oral lichen planus, the concentration of salivary fibronectin is lower than usual . Fibronectin quantity also varies in the presence of some tumours, such as oral squamous cell carcinoma, although it cannot be considered a specific factor . Aims: Due to the low Fn concentration in saliva and its lability in the soluble form, sample collection and conservation conditions are extremely important . The aim of this study is therefore to standardise these conditions so that the Fn can be quantified in an optimum manner . Materials and methods: The Fn concentration in human saliva was determined in 20 healthy subjects aged between 28 and 54 by means of the ELISA technique and the concentration of the protein in fresh samples kept at 4 grades C for 24 hours was compared with that of frozen samples kept at 40 grades C below 0, for different periods of time . Results and Conclusions: After comparing different ways of conserving the saliva samples, we found that the optimum conditions were to collect the samples in glass tubes and to quantify them immediately after collection or conserve them at 4 grades C and quantify them within a maximum of 24 hours . Freezing and later thawing for quantification induced losses of up to 60% of the protein. Int J Colorectal Dis, 2004 Jul, 19(4), 334 - 42 Epub 2004 Apr 22. Activation of NF-kappaB in