J Immunol, 2003 Jul 15, 171(2), 971 - 8 Colitogenic Th1 cells are present in the antigen-experienced T cell pool in normal mice: control by CD4+ regulatory T cells and IL-10; Asseman C et al.; CD4(+) regulatory T cells have been shown to prevent intestinal inflammation; however, it is not known whether they act to prevent the priming of colitogenic T cells or actively control these cells as part of the memory T cell pool . In this study, we describe the presence of colitogenic Th1 cells within the CD4(+)CD45RB(low) population . These pathogenic cells enrich within the CD25(-) subset and are not recent thymic emigrants . CD4(+)CD45RB(low) cells from germfree mice were significantly reduced in their ability to transfer colitis to immune deficient recipients, suggesting the presence of commensal bacteria in the donor mice drives colitogenic T cells into the Ag-experienced/memory T cell pool . This potentially pathogenic population of Ag-experienced T cells is subject to T cell-mediated regulation in vivo by both CD4(+)CD25(+) and CD4(+)CD25(-) cells in an IL-10-dependent manner . Furthermore, administration of an anti-IL-10R mAb to unmanipulated adult mice was sufficient to induce the development of colitis . Taken together, these data indicate that colitogenic Th1 cells enter into the Ag-experienced pool in normal mice, but that their function is controlled by regulatory T cells and IL-10 . Interestingly, IL-10 was not absolutely required for CD4(+)CD25(+) T cell-mediated inhibition of colitis induced by transfer of naive CD4(+)CD45RB(high) cells, suggesting a differential requirement for IL-10 in the regulation of naive and Ag-experienced T cells. J Immunol, 2003 Jul 15, 171(2), 924 - 30 Identification of uteroglobin-related protein 1 and macrophage scavenger receptor with collagenous structure as a lung-specific ligand-receptor pair; Bin LH et al.; High in normal (HIN)-1 is a secreted protein highly expressed in normal breast epithelium and down-regulated in breast carcinomas . By searching GenBank expressed sequence tag databases, we identified HIN-2, a protein homologous to HIN-1 . HIN-2 is identical with a recently identified protein called uteroglobin-related protein 1 (UGRP1) . Northern blot analysis demonstrated that UGRP1 is specifically expressed by lung, but not by the other tissues examined . By in situ hybridization experiments, UGRP1 was shown to be expressed by lung Clara-like cells in the bronchial epithelium and to be up-regulated in cystic fibrosis . In a mammalian expression system, secreted recombinant UGRP1 was copurified with apolipoprotein A-I . Using a retroviral vector-mediated expression cloning approach, we identified macrophage scavenger receptor with collagenous structure (MARCO) as a receptor for UGRP1 . Northern blot and in situ hybridization experiments indicated that MARCO is expressed by alveolar macrophages in the lung . UGRP1 also bound to bacteria and yeast . LPS, a previously identified MARCO ligand, competed with UGRP1 for binding to MARCO and bacteria . Our findings suggest that UGRP1-MARCO is a ligand-receptor pair that is probably involved in inflammation and pathogen clearance in the lung. J Immunol, 2003 Jul 15, 171(2), 836 - 44 Hyperconservation of the N-formyl peptide binding site of M3: evidence that M3 is an old eutherian molecule with conserved recognition of a pathogen-associated molecular pattern; Doyle CK et al.; The mouse MHC class I-b molecule H2-M3 has unique specificity for N-formyl peptides, derived from bacteria (and mitochondria), and is thus a pathogen-associated molecular pattern recognition receptor (PRR) . To test whether M3 was selected for this PRR function, we studied M3 sequences from diverse murid species of murine genera Mus, Rattus, Apodemus, Diplothrix, Hybomys, Mastomys, and Tokudaia and of sigmodontine genera Sigmodon and PEROMYSCUS: We found that M3 is highly conserved, and the 10 residues coordinating the N-formyl group are almost invariant . The ratio of nonsynonymous and synonymous substitution rates suggests the Ag recognition site of M3, unlike the Ag recognition site of class I-a molecules, is under strong negative (purifying) selection and has been for at least 50-65 million years . Consistent with this, M3 alpha1alpha2 domains from Rattus norvegicus and Sigmodon hispidus and from the "null" allele H2-M3(b) specifically bound N-formyl peptides . The pattern of nucleotide substitution in M3 suggests M3 arose rapidly from murid I-a precursors by an evolutionary leap ("saltation"), perhaps involving intense selective pressure from bacterial pathogens . Alternatively, M3 arose more slowly but prior to the radiation of eutherian (placental) mammals . Older dates for the emergence of M3, and the accepted antiquity of CD1, suggest that primordial class I MHC molecules could have evolved originally as monomorphic PRR, presenting pathogen-associated molecular patterns . Such MHC PRR molecules could have been preadaptations for the evolution of acquired immunity during the early vertebrate radiation. J Immunol, 2003 Jul 15, 171(2), 795 - 801 Innate immunity together with duration of antigen persistence regulate effector T cell induction; Storni T et al.; Proliferation of T cells is important for the expansion of specific T cell clones during immune responses . In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells . Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation . Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization . However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs . The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity . In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses . Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses. Eur J Biochem, 2003 Jul, 270(14), 3036 - 46 Structure of the exceptionally large nonrepetitive carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-82164; Vinogradov E et al.; The structures of the oligosaccharides obtained after acetic acid hydrolysis and alkaline deacylation of the rough-type lipopolysaccharide (LPS) from Pectinatus frisingensis strain VTT E-82164 were analysed using NMR spectroscopy, MS and chemical methods . The LPS contains two major structural variants, differing by a decasaccharide fragment, and some minor variants lacking the terminal glucose residue . The largest structure of the carbohydrate backbone of the LPS that could be deduced from experimental results consists of 25 monosaccharides (including the previously found Ara4NP residue in lipid A) arranged in a well-defined nonrepetitive structure: We presume that the shorter variant with R1 = H represents the core-lipid A part of the LPS, and the additional fragment is present instead of the O-specific polysaccharide . Structures of this type have not been previously described . Analysis of the deacylation products obtained from the LPS of the smooth strain, VTT E-79100T, showed that it contains a very similar core but with one different glycosidic linkage. Brief Bioinform, 2003 Jun, 4(2), 133 - 49 Comparative genomics tools applied to bioterrorism defence; Slezak T et al.; Rapid advances in the genomic sequencing of bacteria and viruses over the past few years have made it possible to consider sequencing the genomes of all pathogens that affect humans and the crops and livestock upon which our lives depend . Recent events make it imperative that full genome sequencing be accomplished as soon as possible for pathogens that could be used as weapons of mass destruction or disruption . This sequence information must be exploited to provide rapid and accurate diagnostics to identify pathogens and distinguish them from harmless near-neighbours and hoaxes . The Chem-Bio Non-Proliferation (CBNP) programme of the US Department of Energy (DOE) began a large-scale effort of pathogen detection in early 2000 when it was announced that the DOE would be providing bio-security at the 2002 Winter Olympic Games in Salt Lake City, Utah . Our team at the Lawrence Livermore National Lab (LLNL) was given the task of developing reliable and validated assays for a number of the most likely bioterrorist agents . The short timeline led us to devise a novel system that utilised whole-genome comparison methods to rapidly focus on parts of the pathogen genomes that had a high probability of being unique . Assays developed with this approach have been validated by the Centers for Disease Control (CDC) . They were used at the 2002 Winter Olympics, have entered the public health system, and have been in continual use for non-publicised aspects of homeland defence since autumn 2001 . Assays have been developed for all major threat list agents for which adequate genomic sequence is available, as well as for other pathogens requested by various government agencies . Collaborations with comparative genomics algorithm developers have enabled our LLNL team to make major advances in pathogen detection, since many of the existing tools simply did not scale well enough to be of practical use for this application . It is hoped that a discussion of a real-life practical application of comparative genomics algorithms may help spur algorithm developers to tackle some of the many remaining problems that need to be addressed . Solutions to these problems will advance a wide range of biological disciplines, only one of which is pathogen detection . For example, exploration in evolution and phylogenetics, annotating gene coding regions, predicting and understanding gene function and regulation, and untangling gene networks all rely on tools for aligning multiple sequences, detecting gene rearrangements and duplications, and visualising genomic data . Two key problems currently needing improved solutions are: (1) aligning incomplete, fragmentary sequence (eg draft genome contigs or arbitrary genome regions) with both complete genomes and other fragmentary sequences; and (2) ordering, aligning and visualising non-colinear gene rearrangements and inversions in addition to the colinear alignments handled by current tools. Brief Bioinform, 2003 Jun, 4(2), 105 - 23 An applications-focused review of comparative genomics tools: capabilities, limitations and future challenges; Chain P et al.; A team at the Lawrence Livermore National Laboratory (LLNL) was given the task of using computational tools to speed up the development of DNA diagnostics for pathogen detection . This work will be described in another paper in this issue (see pages 133-149) . To achieve this goal it was necessary to understand the merits and limitations of the various available comparative genomics tools . A review of some recent tools for multisequence/genome alignment and substring comparison is presented, within the general framework of applicability to a large-scale application . We note that genome alignments are important for many things, only one of which is pathogen detection . Understanding gene function, gene regulation, gene networks, phylogenetic studies and other aspects of evolution all depend on accurate nucleic acid and protein sequence alignment . Selecting appropriate tools can make a large difference in the quality of results obtained and the effort required. Pest Manag Sci, 2003 Jun-Jul, 59(6-7), 708 - 17 United States Department of Agriculture-Agricultural Research Service research on natural products for pest management; Duke SO et al.; Recent research of the Agricultural Research Service of USDA on the use of natural products to manage pests is summarized . Studies of the use of both phytochemicals and diatomaceous earth to manage insect pests are discussed . Chemically characterized compounds, such as a saponin from pepper (Capsicum frutescens L), benzaldehyde, chitosan and 2-deoxy-D-glucose are being studied as natural fungicides . Resin glycosides for pathogen resistance in sweet potato and residues of semi-tropical leguminous plants for nematode control are also under investigation . Bioassay-guided isolation of compounds with potential use as herbicides or herbicide leads is underway at several locations . New natural phytotoxin molecular target sites (asparagine synthetase and fructose-1,6-bisphosphate aldolase) have been discovered . Weed control in sweet potato and rice by allelopathy is under investigation . Molecular approaches to enhance allelopathy in sorghum are also being undertaken . The genes for polyketide synthases involved in production of pesticidal polyketide compounds in fungi are found to provide clues for pesticide discovery . Gene expression profiles in response to fungicides and herbicides are being generated as tools to understand more fully the mode of action and to rapidly determine the molecular target site of new, natural fungicides and herbicides. Pest Manag Sci, 2003 Jun-Jul, 59(6-7), 654 - 64 United States Department of Agriculture-Agricultural Research Service research programs in biological control of plant diseases; Roberts DP et al.; A number of USDA-ARS programs directed at overcoming impediments to the use of biocontrol agents on a commercial scale are described . These include improvements in screening techniques, taxonomic studies to identify beneficial strains more precisely, and studies on various aspects of the large-scale production of biocontrol agents . Another broad area of studies covers the ecological aspects of biocontrol agents-their interaction with the pathogen, with the plant and with other aspects of the environmental complex . Examples of these studies are given and their relevance to the further development and expansion of biocontrol agents is discussed. J Br Menopause Soc, 2003 Jun, 9(2), 69 - 74 Probiotics and prebiotics in female health; Smejkal C et al.; Functional foods such as probiotics, prebiotics and nutraceuticals are of extreme interest to researchers . There is growing evidence that these food ingredients may improve and in some cases treat certain conditions that are implicated in women's health . The use of probiotics (live, beneficial bacteria) in improving gastrointestinal and non-gastrointestinal tract conditions such as irritable bowel syndrome, candidiasis and other female urogenital tract conditions are reviewed . Emphasis is also given to the importance of prebiotics (non-digestible food ingredients) in osteoporosis management and alleviation of menopausal symptoms and reducing the onset of cancer. Ann Pathol, 2003 Apr, 23(2), 161 - 4 {An oral infection not to be underestimated: actinomycosis . A study of 4 cases with extensive bone necrosis}; Musso S et al.; We report 4 cases of osteitis with extensive necrosis of the maxillofacial bone due to actinomycosis . Histological study showed soft tissue necrosis with bone involvement, suppuration and granulomatous inflammation . At contact with bone destruction, numerous Gram and PAS positive bacteria with branched filaments, inconsistently associated with granules, were noted . Cultures were negative . Ultrastructural study showed in two cases, some elongated bacteria, variable in size, less to one micron in diameter, associated with ossein destruction . Actinomycetes usually represented in the normal oral flora, may become pathogenic in debilited host . Histologic study is necessary for the diagnosis allowing to confirm the tissue invasion by the bacteria. J Med Genet, 2003 Jul, 40(7), 479 - 86 Subcellular localisation, secretion, and post-translational processing of normal cochlin, and of mutants causing the sensorineural deafness and vestibular disorder, DFNA9; Robertson NG et al.; Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9 . To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown . We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin . By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein . Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments . We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of approximately 50 kDa . Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain . We have also shown that cochlin is N-glycosylated in its mature secreted form . Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain . Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity . To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins . We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation . These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain . It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder. J Clin Microbiol, 2003 Jul, 41(7), 3327 - 30 Detection of Legionella pneumophila by real-time PCR for the mip gene; Wilson DA et al.; A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L . pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria . Eight culture-positive and 40 culture-negative clinical specimens were tested . This assay was 100% sensitive and 100% specific for L . pneumophila. J Biol Chem, 2003 Sep 12, 278(37), 35231 - 40 Epub 2003 Jul 03. Hsp90 inhibition accelerates cell lysis . Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events; Sreedhar AS et al.; The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells . Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure . Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis . The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol . Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors . Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity . When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed . Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria . Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity . Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells. J Microbiol Methods, 2003 Sep, 54(3), 427 - 31 Evidence for chimeric sequences formed during random arbitrarily primed PCR; Chang IS et al.; Chimeric sequences were observed to occur abundantly (48% of clones) during random arbitrarily primed polymerase chain reaction (RAP-PCR) experiments designed to examine differential expression of genes involved in metal resistance in sulfate-reducing bacteria (SRB) . Some of the chimeric sequences were composed of sequence from a gene differentially expressed under the imposed conditions and a sequence of the 16S or 23S rRNA gene . The remainder were composed of two rRNA sequences . Experiments using PCR and genomic sequence analysis showed that the chimeric sequences were not due to a genetic mutation (e.g., recombination, transposition) . As RAP-PCR has been widely used to identify differentially expressed genes, this observation may aid in our interpretation of RAP-PCR data. J Microbiol Methods, 2003 Sep, 54(3), 295 - 313 Experimental models of pulmonary infection; Bakker-Woudenberg IA; Experimental models of pulmonary infection are being discussed, focused on various aspects of good experimental design, such as choice of animal species and infecting strain, and route of infection/inoculation techniques (intranasal inoculation, aerosol inoculation, and direct instillation into the lower respiratory tract) . In addition, parameters to monitor pulmonary infection are being reviewed such as general clinical signs, pulmonary-associated signs, complication of the pulmonary infection, mortality rate, and parameters after dissection of animals . Examples of pulmonary infection models caused by bacteria, fungi, viruses or parasites in experimental animals with intact or impaired host defense mechanisms are shortly summarized including key-references. Eur J Gastroenterol Hepatol, 2003 Jun, 15(6), 593 - 7 Lessons to be learned from the NOD2 gene in Crohn's disease; Hugot JP et al.; The recent discovery that CARD15/NOD2 is involved in the genetic predisposition to Crohn's disease (CD) provides the final demonstration that CD is a genetic disorder . The gene explains about 20% of the genetic susceptibility . CARD15 mutations are present in 30-50% of CD patients compared to 7-20% of healthy controls . Interestingly, CD patients often carry mutations on their two chromosomes suggesting a mutation dose effect . Unfortunately, even if the association between the three main CARD15 mutations (R702W, G908R and 1007fs) and CD is clearly established, it is not useful today to genotype asymptomatic at risk people or inflammatory bowel disease patients as a routine . More interestingly and for the first time, CARD15 points out a specific pathway involved in CD mechanism . Because CARD15 is able to be activated by components of the bacterial wall and further induce the activation of NFkappaB, a proinflammatory molecule, CARD15 discovery makes the link, at the molecular level, between bacteria and inflammation of the digestive tract. J Cell Sci, 2003 Aug 15, 116(Pt 16), 3387 - 97 Epub 2003 Jul 02. Superoxide signalling required for multicellular development of Dictyostelium; Bloomfield G et al.; Reactive oxygen species are known to have a signalling role in many organisms . In bacteria and yeast various response systems have evolved to combat oxidative stress which are triggered by reactive oxygen species . Mammals and plants are known to actively generate reactive oxygen species such as superoxide during signalling responses to a variety of extracellular factors . We report here the generation of superoxide as a signalling molecule in early development of Dictyostelium discoideum . Dictyostelium grows as single amoebae but, on starvation, the single cells aggregate to form a multicellular organism . Superoxide is generated in response to a secreted factor during the transition to the multicellular phase of development . Scavenging superoxide, either pharmacologically or by overexpressing the enzyme superoxide dismutase, inhibits the formation of the aggregate . This report of the use of superoxide as a signalling molecule in a lower eukaryote as it switches to a multicellular phase suggests that this signalling mechanism arose early in the evolution of multicellular organisms, perhaps as a necessary consequence of the need to diversify the number and type of signalling pathways available to facilitate intercellular communication. Genome Res, 2003 Jul, 13(7), 1563 - 71 An evolutionarily structured universe of protein architecture; Caetano-Anolles G et al.; Protein structural diversity encompasses a finite set of architectural designs . Embedded in these topologies are evolutionary histories that we here uncover using cladistic principles and measurements of protein-fold usage and sharing . The reconstructed phylogenies are inherently rooted and depict histories of protein and proteome diversification . Proteome phylogenies showed two monophyletic sister-groups delimiting Bacteria and Archaea, and a topology rooted in Eucarya . This suggests three dramatic evolutionary events and a common ancestor with a eukaryotic-like, gene-rich, and relatively modern organization . Conversely, a general phylogeny of protein architectures showed that structural classes of globular proteins appeared early in evolution and in defined order, the alpha/beta class being the first . Although most ancestral folds shared a common architecture of barrels or interleaved beta-sheets and alpha-helices, many were clearly derived, such as polyhedral folds in the all-alpha class and beta-sandwiches, beta-propellers, and beta-prisms in all-beta proteins . We also describe transformation pathways of architectures that are prevalently used in nature . For example, beta-barrels with increased curl and stagger were favored evolutionary outcomes in the all-beta class . Interestingly, we found cases where structural change followed the alpha-to-beta tendency uncovered in the tree of architectures . Lastly, we traced the total number of enzymatic functions associated with folds in the trees and show that there is a general link between structure and enzymatic function. Appl Environ Microbiol, 2003 Jul, 69(7), 4057 - 66 Toxicity of Al to Desulfovibrio desulfuricans; Amonette JE et al.; The toxicity of Al to Desulfovibrio desulfuricans G20 was assessed over a period of 8 weeks in a modified lactate C medium buffered at four initial pHs (5.0, 6.5, 7.2, and 8.3) and treated with five levels of added Al (0, 0.01, 0.1, 1.0, and 10 mM) . At pH 5, cell population densities decreased significantly and any effect of Al was negligible compared to that of the pH . At pHs 6.5 and 7.2, the cell population densities increased by 30-fold during the first few days and then remained stable for soluble-Al concentrations of <5 x 10(-5) M . In treatments having total-Al concentrations of > or =1 mM, soluble-Al concentrations exceeded 5 x 10(-5) M and limited cell population growth substantially and proportionally . At pH 8.3, soluble-Al concentrations were below the 5 x 10(-5) M toxicity threshold and cell population density increases of 20- to 40-fold were observed . An apparent cell population response to added Al at pH 8.3 was attributed to the presence of large, spirilloidal bacteria (accounting for as much as 80% of the cells at the 10 mM added Al level) . Calculations of soluble-Al speciation for the pH 6.5 and 7.2 treatments that showed Al toxicity suggested the possible presence of the Al(13)O(4)(OH)(24)(H(2)O)(12)(7+) "tridecamer" cation and an inverse correlation of the tridecamer concentration and the cell population density . Analysis by (27)Al nuclear magnetic resonance spectroscopy, however, yielded no evidence of this species in freshly prepared samples or those taken 800 days after inoculation . Exclusion of the tridecamer species from the aqueous speciation calculations at pHs 6.5 and 7.2 yielded inverse correlations of the neutral Al(OH)(3) and anionic Al(OH)(4)(-) monomeric species with cell population density, suggesting that one or both of these ions bear primary responsibility for the toxicity observed. Appl Environ Microbiol, 2003 Jul, 69(7), 3739 - 50 Characterization and in situ carbon metabolism of phototrophic consortia; Glaeser J et al.; A dense population of the phototrophic consortium "Pelochromatium roseum" was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany) . Fluorescence in situ hybridization revealed that the brown epibionts of "P . roseum" constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline . Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low . The 465-bp 16S rRNA gene sequence of the epibiont of "P . roseum" was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR . The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria . Maximum light-dependent H(14)CO(3)(-) fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria . The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (delta(13)C) of their specific biomarkers . Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline . Unexpectedly, isorenieratene and beta-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent . Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their delta(13)C values were determined . Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in (13)C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria . The difference between the delta(13)C values of farnesol, the major esterifying alcohol of BChl e, and CO(2) was -7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of "P . roseum" in situ is photoautotrophic. Infect Dis Obstet Gynecol, 2003, 11(1), 19 - 26 Leukocyte esterase activity in vaginal fluid of pregnant and non-pregnant women with vaginitis/vaginosis and in controls; Mardh PA et al.; OBJECTIVES: To determine the leukocyte esterase (LE) activity in vaginal lavage fluid of women with acute and recurrent vulvovaginal candidosis (VVC and RVVC respectively), bacterial vaginosis (BV), and in pregnant and non-pregnant women without evidence of the three conditions . Also to compare the result of LE tests in women consulting at different weeks in the cycle and trimesters of pregnancy . The LE activity was correlated to vaginal pH, number of inflammatory cells in stained vaginal smears, type of predominating vaginal bacteria and presence of yeast morphotypes . METHODS: One hundred and thirteen women with a history of RVVC, i.e . with at least four attacks of the condition during the previous year and who had consulted with an assumed new attack of the condition, were studied . Furthermore, we studied 16 women with VVC, 15 women with BV, and 27 women attending for control of cytological abnormalities, who all presented without evidence of either vaginitis or vaginosis . Finally, 73 pregnant women were investigated . The LE activity in vaginal fluid during different weeks in the cycle of 53 of the women was measured . RESULTS: In the non-pregnant women, an increased LE activity was found in 96, 88, 73 and 56% of those with RVVC, VVC and BV and in the non-VVC/BV cases, respectively . In 73% of pregnant women in the second trimester, and 76% of those in the third, the LE test was positive . In all groups of non-pregnant women tested, the LE activity correlated with the number of leukocytes in vaginal smears, but it did not in those who were pregnant . There was no correlation between LE activity and week in cycle . The vaginal pH showed no correlation to LE activity in any of the groups studied . CONCLUSIONS: The use of commercial LE dipsticks has a limited value in the differential diagnosis of RVVC, VVC and BV . There is no correlation between the LE activity in vaginal secretion on one hand and vaginal pH, week in the menstrual cycle and trimester in pregnancy on the other . Women with BV often have signs of inflammation as evidenced by a positive LE test and inflammatory cells in genital smears. Nurs Times, 2003 Jun 10-16, 99(23), 63 - 4 The physiology of mucus and sputum production in the respiratory system; Richardson M; The main function of the respiratory system is to draw air into the lungs to allow the exchange of gases with blood circulating to the lungs . This blood supplies the cells of the body with oxygen and removes the waste products of metabolism . Tissues of the respiratory tract are thin and delicate, and become thinnest at the surfaces of the aveoli, where gaseous exchange occurs . The body has a number of mechanisms which protect these tissues and ensure that debris and bacteria do not reach them. Pediatrics, 2003 Jul, 112(1 Pt 1), 87 - 95 Mycoplasma disease and acute chest syndrome in sickle cell disease; Neumayr L et al.; BACKGROUND: Acute chest syndrome (ACS) is the leading cause of hospitalization, morbidity, and mortality in patients with sickle cell disease . Radiographic and clinical findings in ACS resemble pneumonia; however, etiologies other than infectious pathogens have been implicated, including pulmonary fat embolism (PFE) and infarction of segments of the pulmonary vasculature . The National Acute Chest Syndrome Study Group was designed to identify the etiologic agents and clinical outcomes associated with this syndrome . METHODS: Data were analyzed from the prospective study of 671 episodes of ACS in 538 patients with sickle cell anemia . ACS was defined as a new pulmonary infiltrate involving at least 1 complete segment of the lung, excluding atelectasis . In addition, the patients had to have chest pain, fever >38.5C, tachypnea, wheezing, or cough . Samples of blood and deep sputum were analyzed for evidence of bacteria, viruses, and PFE . Mycoplasma pneumoniae infection was determined by analysis of paired serologies . Detailed information on patient characteristics, presenting signs and symptoms, treatment, and clinical outcome were collected . RESULTS: Fifty-one (9%) of 598 episodes of ACS had serologic evidence of M pneumoniae infection . Twelve percent of the 112 episodes of ACS occurring in patients younger than 5 years were associated with M pneumoniae infection . At the time of diagnosis, 98% of all patients with M pneumoniae infection had fever, 78% had a cough, and 51% were tachypneic . More than 50% developed multilobar infiltrates and effusions, 82% were transfused, and 6% required assisted ventilation . The average hospital stay was 10 days . Evidence of PFE with M pneumoniae infection was seen in 5 (20%) of 25 patients with adequate deep respiratory samples for the PFE assay . M pneumoniae and Chlamydia pneumoniae was found in 16% of patients with diagnostic studies for C pneumoniae . Mycoplasma hominis was cultured in 10 (2%) of 555 episodes of ACS and occurred more frequently in older patients, but the presenting symptoms and clinical course was similar to those with M pneumoniae . CONCLUSIONS: M pneumoniae is commonly associated with the ACS in patients with sickle cell anemia and occurs in very young children . M hominis should be considered in the differential diagnosis of ACS . Aggressive treatment with broad-spectrum antibiotics, including 1 from the macrolide class, is recommended for all patients as well as bronchodilator therapy, early transfusion, and respiratory support when clinically indicated. J Bacteriol, 2003 Jul, 185(14), 4186 - 94 Structural analysis of the domain interface in DrrB, a response regulator of the OmpR/PhoB subfamily; Robinson VL et al.; The N-terminal regulatory domains of bacterial response regulator proteins catalyze phosphoryl transfer and function as phosphorylation-dependent regulatory switches to control the output activities of C-terminal effector domains . Structures of numerous isolated regulatory and effector domains have been determined . However, a detailed understanding of regulatory interactions among these domains has been limited by the relative paucity of structural data for intact multidomain response regulator proteins . The first multidomain structures determined, those of transcription factor NarL and methylesterase CheB, both revealed extensive interdomain interfaces . The regulatory domains obstruct access to the functional sites of the effector domains, indicating a regulatory mechanism based on inhibition . In contrast, the recently determined structure of the OmpR/PhoB homologue DrrD revealed no significant interdomain interface, suggesting that the domains are tethered by a flexible linker and lack a fixed orientation relative to each other . To address the generality of this feature, we have determined the 1.8-A resolution crystal structure of Thermotoga maritima DrrB, providing a second structure of a multidomain response regulator of the OmpR/PhoB subfamily . The structure reveals an extensive domain interface of 751 A(2) and therefore differs greatly from that observed in DrrD . Residues that are crucial players in defining the activation state of the regulatory domain contribute to this interface, implying that conformational changes associated with phosphorylation will influence these intramolecular contacts . The DrrB and DrrD structures are suggestive of different signaling mechanisms, with intramolecular communication between N- and C-terminal domains making substantially different contributions to effector domain regulation in individual members of the OmpR/PhoB family. J Am Chem Soc, 2003 Jul 9, 125(27), 8186 - 94 Photoisomerization and proton transfer in photoactive yellow protein; Thompson MJ et al.; The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes . Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates . The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates . The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters . The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al . {Genick, U . K.; Soltis, S . M.; Kuhn, P.; Canestrelli, I . L.; Getzoff, E . D . Nature 1998, 392, 206-209} supports its assignment to the PYP(B) (I(0)) intermediate . Differences between the time-resolved room temperature (298 K) spectrum of the PYP(B) intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter . This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP(L) (I(1) or pR) intermediate . The structure of the chromophore in the PYP(L) intermediate is determined computationally and shown to be deprotonated, in agreement with experiment . Calculations based on our PYP(B) and PYP(L) models lead to insights concerning the PYP(BL) intermediate, observed only at low temperature . The results suggest that the proton is more mobile between Glu46 and the chromophore than previously realized . The findings presented here provide an example of the insights that theoretical studies can contribute to a unified analysis of experimental structures and spectra. Nucleic Acids Res Suppl, 2001, (1), 251 - 2 Studies on the molecular mechanism of the interactions between the cyanobacterial transcription factor, SmtB, and its recognition DNA sequences; Morita EH et al.; Tolerance to the heavy-metal ion stress in cyanobacterial cell is regulated by the transcriptional repressor SmtB that senses the Zn/Cd concentration in the cell . SmtB regulated the transcriptional level of class II metallothionein, SmtA . There are two recognition DNA sequences (Bbs1 and Bbs2) in the operator/promoter region of smtA gene . To clear the functional meaning of the presence of these two sequences, we have compared the affinities of native and point-mutated SmtBs to these two sites . We also have compared the sizes of the protein-DNA complexes being formed with these two sites . SmtB forms protein-DNA complex in an unique size with Bbs1, in three different sizes with Bbs2 . We have further designed the modified Bbs1 and Bbs2 sequences, and the results obtained with these sequences indicate that the differences observed between the cases of Bbs1 and Bbs2 are caused by the presence of direct repeat sequence and the differences in the linker sequences. Zentralbl Gynakol, 2003 Feb, 125(2), 35 - 7 {Mastitis puerperalis}; Rogmans G; Mastitis puerperalis may result either from a blocked mastitis or through bacteria . In rare cases it can originate from a candida infection . Physical measures are initially taken to treat blocked mastitis . Treatment for bacterial mastitis depends upon the expected range of pathogenes, and is therefore primarily treated with cephalosporides . For candida infections, nystantin is the first choice of treatment . Where conservative treatment for suspected mastitis does not lead to an improvement within 24 hours, antibiotics must necessarily be introduced . If, despite these measure, an abscess begins to form, this can be punctured if the patient is protected by antibiotics . Surgical intervention is only necessary in exceptional cases, where the abscess needs to be split open and a loop fitted . Bromocriptin is not suitable for treating mastitis if the mother wishes to continue breastfeeding. Naturwissenschaften, 2003 Jun, 90(6), 273 - 6 Epub 2003 May 07. Rotifers colonising sediments with shallow gas hydrates; Sommer S et al.; Rotifers, one of the smallest metazoans, are only seldom found in marine environments . Surprisingly, we discovered high abundances of at least two new species of rotifers settling in anoxic and highly sulphidic sediments associated with shallow gas hydrates (GH) at the southern crest of Hydrate Ridge off Oregon, NE Pacific, in a water depth of about 780 m . At basins adjacent to Hydrate Ridge, 1,285-2,304 m deep, we found rotifers co-occurring with the sulphide-oxidising bacteria Thioploca sp. Br Dent J, 2003 May 10, 194(9), 485 - 8 Periodontal disease incidence, progression and rate of tooth loss in a general dental practice: the results of a 12-year retrospective analysis of patient's clinical records; Nicholls C; One of the main objectives of a general dental practice is to teach its patients how to reduce their oral bacterial load . In other words, by teaching good oral hygiene habits, it is possible to reduce the number of bacteria and hence the number of species of bacteria present in each patient's mouth . Thus by achieving a high level of oral hygiene, the dental team hope to reduce each patient's future risk of periodontal problems, and dental caries. J Biol Chem, 2003 Sep 12, 278(37), 35542 - 51 Epub 2003 Jun 30. Probing the orientation of reconstituted maltoporin channels at the single-protein level; Danelon C et al.; Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding . This asymmetry revealed also that maltoporin insertion is directional . However, the orientation in the lipid bilayer remained an open question . To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition . In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side . In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition . Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions . We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side. J Clin Periodontol, 2003 Jul, 30(7), 616 - 23 Delayed neutrophil apoptosis in chronic periodontitis patients; Gamonal J et al.; BACKGROUND, AIMS: Neutrophil cells constitute the first defense barrier against the oral bacterial challenge in the periodontium . Reduction of neutrophils could impair this response against periopathogenic bacteria such as Porphyromonas gingivalis . Our previous work implicates the apoptosis of neutrophils in the pathogenesis of periodontitis . We now demonstrate that granulocyte monocyte-colony stimulating factor (GM-CSF) present in the gingival crevicular fluid (GCF) and secreted during the immune response reduces the apoptosis of neutrophils . METHOD: In this study, the presence of GM-CSF and tumor necrosis factor-alpha (TNF-alpha) in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva . GCF was collected for 30 s using Periopaper(R) strips, and cytokines were quantified by ELISA . We used ex vivo culture of gingival tissue biopsies for 2 and 4 days in the presence of GM-CSF . Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique, and expression of Bax by immunohistochemistry . RESULTS: The presence of GM-CSF and TNF-alpha was detected in the majority of sites from periodontal patients (83.3% and 63.3%, respectively), presenting a total amount of 27.65 and 42.38 pg, respectively . GM-CSF reduces the neutrophil apoptosis determined by double staining with TUNEL and myeloperoxidase and by a reduction of Bax expression . CONCLUSIONS: These findings suggest a novel mechanism by which neutrophils specifically accumulate in adult patients with periodontitis. Proteins, 2003 Aug 1, 52(2), 254 - 62 Molecular dynamics simulations of a helicase; Cox K et al.; Helicases are ubiquitous enzymes involved in nucleic acid metabolism . The PcrA DNA helicase is an essential bacterial protein involved in rolling circle plasmid replication and DNA repair . Recent crystal structures of PcrA bound to DNA indicate that a flexible loop mediates a functionally important rigid-body-domain rotation . In this study, we report stochastic boundary molecular dynamics simulations focused on this region for wild-type and mutants designed to increase the rigidity of the region . Residues in the region that were helix-disfavoring, such as glycine, threonine, and others, were mutated to alanine . The simulated dynamics, analyzed with a variety of measures of structure and mobility, indicate that a few point mutations will substantially increase helix formation in this region . Subnanosecond stochastic boundary molecular dynamics simulations at several temperatures offer a rapid protocol for assessing large numbers of mutants and provides a novel strategy for the design of experiments to test the role of this flexible loop region in the function of PcrA . Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1219 - 23 Epub 2003 Jun 27. Structure of the hypothetical protein AQ_1354 from Aquifex aeolicus; Oganesyan V et al.; The crystal structure of a hypothetical protein AQ_1354 (gi 2983779) from the hyperthermophilic bacteria Aquifex aeolicus has been determined using X-ray crystallography . As found in many structural genomics studies, this protein is not associated with any known function based on its amino-acid sequence . PSI-BLAST analysis against a non-redundant sequence database gave 68 similar sequences referred to as 'conserved hypothetical proteins' from the uncharacterized protein family UPF0054 (accession No . PF02310) . Crystallographic analysis revealed that the overall fold of this protein consists of one central alpha-helix surrounded by a four-stranded beta-sheet and four other alpha-helices . Structure-based homology analysis with DALI revealed that the structure has a moderate to good resemblance to metal-dependent proteinases such as collagenases and gelatinases, thus suggesting its possible molecular function . However, experimental tests for collagenase and gelatinase-type function show no detectable activity under standard assay conditions . Therefore, we suggest either that the members of the UPF0054 family have a similar fold but different biochemical functions to those of collagenases and gelatinases or that they have a similar function but perform it under different conditions. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 121 - 8 Use of Bordetella bronchiseptica and Bordetella pertussis as live vaccines and vectors for heterologous antigens; Stevenson A et al.; Bordetella pertussis and Bordetella bronchiseptica are respiratory pathogens of humans and animals respectively . Unlike many bacteria, they are able to efficiently colonise healthy ciliated respiratory mucosa . This characteristic of Bordetella spp . can potentially be exploited to develop efficient live vaccines and vectors for delivery of heterologous antigens to the respiratory tract . Here we review the progress in this area. FEBS Lett, 2003 Jul 10, 546(2-3), 359 - 64 Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins; Goebeler V et al.; Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins . To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system . We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells . Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures . The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM . Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation . These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation. Curr Opin Microbiol, 2003 Jun, 6(3), 229 - 35 Biodesulfurization of fossil fuels; Gray KA et al.; Biotechnological techniques enabling the specific removal of sulfur from fossil fuels have been developed . In the past three years there have been important advances in the elucidation of the mechanisms of biodesulfurization; some of the most significant relate to the role of a flavin reductase, DszD, in the enzymology of desulfurization, and to the use of new tools that enable enzyme enhancement via DNA manipulation to influence both the rate and the substrate range of Dsz . Also, a clearer understanding of the unique desulfinase step in the pathway has begun to emerge. Curr Opin Struct Biol, 2003 Jun, 13(3), 325 - 33 Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P; Harris ME et al.; In bacteria, the tRNA-processing endonuclease ribonuclease P is composed of a large ( approximately 400 nucleotide) catalytic RNA and a smaller ( approximately 100 amino acid) protein subunit that is essential for substrate recognition . Current biochemical and biophysical investigations are providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate specificity and the role of essential metal ions in catalysis . Together with recent high-resolution structures of portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and protein are coordinated in this ribonucleoprotein enzyme. Acta Histochem, 2003, 105(2), 135 - 42 Ionic liquids in embalming and tissue preservation . Can traditional formalin-fixation be replaced safely? Majewski P, Pernak A, Grzymislawski M, Iwanik K, Pernak J. Ionic liquids (ILs) can be used for embalming and tissue preservation . ILs does not cause tissue damage and the tissue colour remains unaltered after treatment . Microscopical morphology of tissues fixed in ILs is of better quality than that of tissues fixed in formalin . Tissue preservation depends on the type of ILs . Best results were obtained with 1-methyl-3-octyloxymethylimidazolium tetrafluoroborate, the density of which resembles that of water . The salt is nonvaporous and when used as a formalin substitute, it eliminates health hazards in the pathological laboratory. Environ Sci Technol, 2003 Jun 1, 37(11), 2323 - 31 Terpenoids as major precursors of dissolved organic matter in landfill leachates, surface water, and groundwater; Leenheer JA et al.; 13C NMR analyses of hydrophobic dissolved organic matter (DOM) fractions isolated from a landfill leachate contaminated groundwater near Norman, OK; the Colorado River aqueduct near Los Angeles, CA; Anaheim Lake, an infiltration basin for the Santa Ana River in Orange County, CA; and groundwater from the Tomago Sand Beds, near Sydney, Australia, found branched methyl groups and quaternary aliphatic carbon structures that are indicative of terpenoid hydrocarbon precursors . Significant amounts of lignin precursors, commonly postulated to be the major source of DOM, were found only in trace quantities by thermochemolysis/gas chromatography/mass spectrometry of the Norman Landfill and Tomago Sand Bed hydrophobic DOM fractions . Electrospray/tandem mass spectrometry of the Tomago Sand Bed hydrophobic acid DOM found an ion series differing by 14 daltons, which is indicative of aliphatic and aryl-aliphatic polycarboxylic acids . The product obtained from ozonation of the resin acid, abietic acid, gave a similar ion series . Terpenoid precursors of DOM are postulated to be derived from resin acid paper sizing agents in the Norman Landfill, algal and bacterial terpenoids in the Colorado River and Anaheim Lake, and terrestrial plant terpenoids in the Tomago Sand Beds. Refuat Hapeh Vehashinayim, 2003 Apr, 20(2), 8 - 23, 78 Dietary determinants of dental caries and dietary recommendations for preschool children; Tinanoff N et al.; OBJECTIVES: The purpose of this review, commissioned by the Administration for Children and Families, the Health Resources and Services Administration, the Health Care Financing Administration, and the Department of Agriculture's Food and Nutrition Service, was to update the evidence of the dietary factors that affect dental caries, and subsequently formulate dietary recommendations for preschool children based on principles of cariology . METHODS: Literature on the dental caries process, dietary factors affecting dental caries initiation and progression, nutrition education and counseling were reviewed and synthesized . Dietary guidelines for children at various ages were then constructed based on the review . RESULTS: Dental caries in preschool children is due to a combination of factors, including colonization of teeth with cariogenic bacteria, type of foods and frequency of exposure of these foods to the cariogenic bacteria, and susceptible teeth . Caries risk is greatest if sugars are consumed at high frequency and are in a form that is retained in the mouth for long periods . Sucrose is the most cariogenic sugar because it can form glucan that enables firm bacterial adhesion to teeth and limits diffusion of acid and buffers in the plaque . There is emerging interest in the effects of tooth development and its role in the future dental caries risk of the child . CONCLUSIONS: Nutrition education and counseling for the purposes of reducing caries in children is aimed at teaching parents the importance of reducing high frequency exposures to obvious and hidden sugars . Guidelines include: avoiding frequent consumption of juice or other sugar containing drinks in bottle or sippy cup; discouraging the behavior of a child sleeping with a bottle; promoting non-cariogenic foods for snacks; fostering eating patterns consistent with Food Guide Pyramid; limiting cariogenic foods to mealtimes; rapidly clearing cariogenic foods from the child's oral cavity either by tooth brushing or by consumption of protective foods; and restricting sugar containing snacks that are slowly eaten (e.g., candy, cough drops, lollipops, suckers) . Along with nutritional factors, a comprehensive approach to preventing dental caries in preschool children must include improved general dietary habits, good oral hygiene, appropriate use of fluorides, and access to preventive and restorative dental care. Clin Infect Dis, 2003 Jul 1, 37(1), 88 - 95 Epub 2003 Jun 25. Progress toward a pathogen-free blood supply; McCullough J; Although the nation's blood supply is safer than ever, a small risk of transfusion-transmitted infection remains . Present strategies to further reduce the risk, such as the donor medical evaluation or laboratory testing, will not likely eliminate this risk . A different approach involves treating donated blood to eliminate its infectivity . A pathogen-inactivated plasma product was available for several years but was recently withdrawn . Several other methods are under development, but all of these prevent nucleic acids from replicating, thus inactivating any contaminating viruses or bacteria . Toxicity, mutagenicity, and safety margins seem to be adequate, and damage to blood proteins or cellular elements is minimal . Clinical trials of pathogen-inactivated platelets have been completed in Europe and in the United States, and phase III clinical trials of pathogen-inactivated red blood cells are underway in the United States . If these encouraging results are sustained, the risk of transfusion-transmitted disease may be nearly eliminated. Carbohydr Res, 2003 Jul 4, 338(14), 1455 - 67 Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase; Borriss R et al.; The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure {beta-D-Glcp-(1-->3)}(n)-beta-D-Glcp-MeUmb, where n=1-5 . The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp . and from a homogenate of the marine mollusc Spisula sachalinensis . Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor . Modification of {beta-D-Glcp-(1-->3)}(2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides . The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry . The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides . When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2). FEMS Microbiol Rev, 2003 Jun, 27(2-3), 165 - 81 Zn, Cu and Co in cyanobacteria: selective control of metal availability; Cavet JS et al.; Homeostatic systems for essential and non-essential metals create the cellular environments in which the correct metals are acquired by metalloproteins while the incorrect ones are somehow avoided . Cyanobacteria have metal requirements often absent from other bacteria; copper in thylakoidal plastocyanin, zinc in carboxysomal carbonic anhydrase, cobalt in cobalamin but magnesium in chlorophyll, molybdenum in heterocystous nitrogenase, manganese in thylakoidal water-splitting oxygen-evolving complex . This article reviews: an intracellular trafficking pathway for inward copper supply, the sequestration of surplus zinc by metallothionein (also present in other bacteria) and the detection and export of excess cobalt . We consider the influence of homeostatic proteins on selective metal availability. FEMS Microbiol Rev, 2003 Jun, 27(2-3), 145 - 63 The MerR family of transcriptional regulators; Brown NL et al.; The MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognised . The signature of the family is amino acid similarity in the first 100 amino acids, including a helix-turn-helix motif followed by a coiled-coil region . With increasing recognition of members of this class over the last decade, particularly with the advent of rapid bacterial genome sequencing, MerR-like regulators have been found in a wide range of bacterial genera, but not yet in archaea or eukaryotes . The few MerR-like regulators that have been studied experimentally have been shown to activate suboptimal sigma(70)-dependent promoters, in which the spacing between the -35 and -10 elements recognised by the sigma factor is greater than the optimal 17+/-1 bp . Activation of transcription is through protein-dependent DNA distortion . The majority of regulators in the family respond to environmental stimuli, such as oxidative stress, heavy metals or antibiotics . A subgroup of the family activates transcription in response to metal ions . This subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region, but it is not yet clear how metal discrimination occurs . This subgroup of MerR family regulators includes MerR itself and may have evolved to generate a variety of specific metal-responsive regulators by fine-tuning the sites of metal recognition. FEBS Lett, 2003 Jul 3, 546(1), 87 - 92 Spectral imaging and its applications in live cell microscopy; Zimmermann T et al.; In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample . However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods . Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging . This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples . Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples. FEBS Lett, 2003 Jul 3, 546(1), 25 - 31 Phosphoinositide signaling disorders in human diseases; Pendaries C et al.; Phosphoinositides (PIs) play an essential role in diverse cellular functions . Their intracellular level is strictly regulated by specific PI kinases, phosphatases and phospholipases . Recent discoveries indicate that dysfunctions in the control of their level often lead to pathologies . This review will focus on some human diseases whose etiologies involve PI-metabolizing enzymes . The role of PTEN (phosphatase and tensin homolog deleted on chromosome ten) in cancer, the impact of the Src homology 2-containing inositol-5-phosphatase phosphatases in acute myeloid leukemia or diabetes, the involvement of myotubularin family members in genetic diseases and the implication of OCRL1 in Lowe syndrome will be emphasized . We will also review how some bacterial pathogens have evolved strategies to specifically manipulate the host cell PI metabolism to efficiently infect them. Methods, 2003 Aug, 30(4), 322 - 9 Making a better RNAi vector for Drosophila: use of intron spacers; Lee YS et al.; Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi) . This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster . Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method . We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing . A single copy of the transgene effectively and uniformly silences expression of a target gene (white) in transgenic flies . We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene . The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4 . The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner . Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure. Methods, 2003 Aug, 30(4), 289 - 95 Constructs and methods for high-throughput gene silencing in plants; Helliwell C et al.; Gene silencing can be achieved by transformation of plants with constructs that express self-complementary (termed hairpin) RNA containing sequences homologous to the target genes . The DNA sequences encoding the self-complementary regions of hairpin (hp) RNA constructs form an inverted repeat . The inverted repeat can be stabilized in bacteria through separation of the self-complementary regions by a "spacer" region . When the spacer sequence encodes an intron, the efficiency of gene silencing is very high . There are at least three ways in which hpRNA constructs can be made . The construct may be generated from standard binary plant transformation vectors in which the hairpin-encoding region is generated de novo for each gene . Alternatively, generic gene-silencing vectors such as the pHANNIBAL and the pHELLSGATE series can be used . They simply require the insertion of PCR products, derived from the target gene, into the vectors by conventional cloning or by using the Gateway directed recombination system . In this article, we describe and evaluate the advantages of these vectors and then provide the protocols for their efficient use. Nat Prod Rep, 2003 Jun, 20(3), 327 - 41 Biosynthesis of chlorophylls from protoporphyrin IX; Willows RD; A review of the biosynthesis of chlorophylls and bacteriochlorophylls from protoporphyrin IX with 235 references . The literature on the enzymes magnesium chelatase, S-adenosyl-L-methionine:magnesium protoporphyrin IX O-methyltransferase, magnesium-protoporphyrin IX monomethyl ester oxidative cyclase, protochlorophyllide oxidoreductase, chlorophyll synthase, bacteriochlorophyll synthase, protochlorophyllide 8-vinyl reductase and chlorophyll a oxidase from 1989 is discussed. Ying Yong Sheng Tai Xue Bao, 2003 Feb, 14(2), 305 - 9 {Effect of inorganic nitrogen on CH4 oxidation in soils}; Wang Z et al.; The effects of N inputs, including N fertilizer applications and atmospheric N depositions, on CH4 oxidation by soils were determined by CH4-oxidizing bacteria, N inputs and soil factors . These effects were inhibitory or stimulative, but inhibition was more popular than stimulation, and the inhibition of NH4+ was more popular than that of NO3- . Wealthy experiments in different soils and climate regions are required to monitor and evaluate how N inputs influence CH4 oxidation in soils . Generally, CH4 oxidation in soils following N inputs showed multiple inhibition patterns, i.e., immediate or direct inhibition, delayed inhibition, and absence of inhibition . Some researchers explained the inhibition mechanism through using competition between CH4 and NH4+ for the same enzyme sites, elevated threshold, salt effect and ion-exchange, and N turnover rate and N concentration . However, the inhibitory mechanism is still unclear . Therefore, inhibitory mechanism is one of the main research concerns in future. Cell Mol Life Sci, 2003 May, 60(5), 919 - 41 Recently discovered functions of glucosylceramides in plants and fungi; Warnecke D et al.; Glycosphingolipids are ubiquitous membrane lipids of eukaryotic organisms and a few bacteria . Whereas inositol-containing glycosphingolipids are restricted to plants and fungi, galactosylceramide occurs only in fungi and animals . In contrast, glucosylceramide is the unique glycosphingolipid which plants, fungi and animals have in common . However, there are specific differences in the structure of the ceramide backbone of glucosylceramides from these organisms . A comparison of the structural features and the biosynthesis of glucosylceramides from plants, fungi and animals will contribute to our understanding of their functions, which so far have been analysed mainly in animals . The availability of nearly all genes involved in the biosynthesis of glucosylceramides enables the specific manipulation of glycosphingolipid metabolism by techniques of forward and reverse genetics . Application of this approach to unicellular organisms like yeasts, multicellular filamentous fungi, as well as to complex organisms like plants will reveal common and different glucosylceramide functions in these organisms . These glycolipids play a role both in intracellular processes and in cell-to-cell interactions . These interactions may occur between cells of a multicellular organism or between cells of different species, as in host-pathogen interactions. Cell Mol Life Sci, 2003 May, 60(5), 854 - 61 Translation repression by antisense sequences; Good L; Antisense sequences that hybridize to messenger RNA can inhibit target gene expression in a variety of ways . The best-known antisense mechanisms trigger messenger RNA (mRNA) degradation; however, translation repression by ribosome interference is a common natural antisense mechanism . In bacteria, there are fascinating examples of cis-encoded and trans-encoded antisense sequences that reversibly repress translation . In eukaryotes, microRNAs that bind 3'UTR target sequences also repress translation, although the mechanism is unclear . An important feature of translation repression is that the mRNA can remain intact during periods of repression, and rapid expression switching can occur in response to cellular and environmental signals . Recent genome analyses indicate many new short noncoding RNAs with predicted antisense activities . Therefore, translation repression by antisense sequences is likely to be a common and important form of posttranscriptional gene control, and such natural mechanisms provide a basis for the development of synthetic antisense gene control in research and drug development. Cell Mol Life Sci, 2003 May, 60(5), 844 - 53 Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues; Gait MJ; Improving the delivery of synthetic oligonucleotides and their analogues into cells is an important goal in the full development of antisense technology for control of gene expression in cell culture and in vivo . This review describes the harnessing of certain peptides, either as noncovalent complexes or as covalent conjugates, to enhance the delivery of antisense oligonucleotides into cells and/or to affect their cell localization . Phosphodiester and phosphorothioate oligonucleotides are included as well as peptide nucleic acids (PNAs), analogues of oligonucleotides where the negatively charged phosphate backbone is replaced by a neutral amide linkage . This review contains a critical evaluation of claims for certain peptide-oligonucleotide conjugates to translocate into cultured cells by a non-energy-dependent nonendosomal route . In addition, the available evidence for the utility of stable versus nonstable linkages between peptide and oligonucleotide or PNA is discussed. Wilderness Environ Med, 2003 Summer, 14(2), 101 - 5 Catfish spine envenomation: a case of delayed presentation; Ajmal N et al.; Catfish spine envenomations can result in debilitating hand problems . Virulent bacteria may be introduced through a puncture wound . An offending organism may be difficult to culture, and a foreign body may be missed unless there is a high index of suspicion . The majority of cases present early and symptoms resolve within 3 months . We report a markedly delayed presentation and treatment of a catfish "finning" injury that resulted in chronic tenosynovitis to the hand . A review of the literature and current treatment recommendations are provided. Scand J Gastroenterol, 2003 Jun, 38(6), 588 - 93 Effect of short-term treatment with regular or high doses of omeprazole on the detection of Helicobacter pylori in bleeding peptic ulcer patients; Udd M et al.; BACKGROUND: It is unknown whether short-term regular or high-dose omeprazole has any influence on the colonization of Helicobacter pylori in the stomach . We therefore studied the effect of 3-day treatment of 2 different doses of omeprazole . METHODS: H . pylori-positive patients with peptic ulcer bleeding (n = 101) were randomized to receive either a regular dose (20 mg/day for 3 days) (n = 51) or a high dose of omeprazole (80 mg bolus + 8 mg/h infusion/day for 3 days) (n = 50) . H . pylori status was assessed by histology and urease testing of gastric biopsies pre-entry and after 3-day therapy . RESULTS: With the high dose of omeprazole, tests for the diagnosis of H . pylori became negative significantly more often than with the regular dose (60% versus 27.5%, P=0.001 (any test), 67.6% versus 31.7%, P=0.003 (histology) and 82.2% versus 43.6%, P=0.001 (urease test)) . CONCLUSION: Conversion of the H . pylori tests negative after 3-day treatment of omeprazole is dose-dependent . The diagnosis of H . pylori infection depends on the timing of biopsies in relation to the beginning of proton-pump inhibitor treatment . If samples to find H . pylori are not taken before the treatment, the presence of the bacteria may be overlooked. Med Sci Monit, 2003 Jun, 9(6), BR225 - 30 Identification of acidic, alkaline, and neutral sphingomyelinase activities in Mycobacterium tuberculosis; Vargas-Villarreal J et al.; BACKGROUND: Sphingomyelinase enzymes are pathogenic factors of several intracellular bacteria species, which have been little studied in Mycobacterium tuberculosis . MATERIAL/METHODS: Cell free extracts from H37Rv and CDC-1551 M . tuberculosis strains were assayed for sphingomyelinase activity by using {N-methyl-14C}-sphingomyelin as substrate . Double-directional thin-layer chromatography was used to separate the substrate and hydrolysis product . Sphingomyelinase activity was analyzed as a function of incubation time, dose, pH and the presence of MgCl2, CaCl2, ZnSO4, HgCl2, MnCl2, CoCl2 and EDTA (1 or 10 mM) . RESULTS: Mycobacterial preparations hydrolyzed {14C}-sphingomyelin, in time- and dose-dependent manners, producing {14C}-phosphorylcholine as a unique product . The activity of H37Rv neutral sphingomyelinase at pH 7.5 was 2.15 times higher than that of CDC-1551 . This activity was inhibited 21-82% by Ca2+, Hg2+ or Zn2+ and EDTA, and stimulated 40-117% by Mn2+ and Mg2+ . In addition, preparations from both strains showed two peaks of sphingomyelinase, one at pH 5.5 and the other at pH 3.0 . However, these activities were 4-22 times lower than that observed at pH 7.5 for strain H37Rv . Preparations from H37Rv, but not those of CDC-1551, hydrolyzed sphingomyelin at pH 8-9, with a specific activity similar to that of the neutral CDC-1551 enzyme . CONCLUSIONS: Both strains H37Rv and CDC-1551 of M . tuberculosis have cation-dependent acidic and neutral sphingomyelinase-C enzymes, showing the neutral as the major activity . In addition, H37Rv has an alkaline sphingomyelinase-C . The importance of SMases in M . tuberculosis pathogenesis remains to be elucidated. Vet Pathol, 2003 Jul, 40(4), 421 - 32 Immunopathogenesis of experimentally induced proliferative enteropathy in pigs; MacIntyre N et al.; To characterize the immune response associated with Lawsonia intracellularis infection, twenty-eight, 7-week-old pigs were dosed orally with a pure culture of L . intracellularis . Animals were killed 3, 7, 14, 21, 28, 35, and 42 days postinfection . Light microscopic studies were undertaken to immunophenotype the immunologic response using specific antibodies to T-cell subsets (CD3, CD4, and CD8), B cells, major histocompatibility complex class II, cadherin, and macrophages over the course of time . The results indicate that there is a direct association between the presence of L . intracellularis and reduced T-cell and B-cell numbers . For the first time, this provides evidence of the presence of an immunosuppressive mechanism operating in this disease . Furthermore, macrophage marker studies indicated that macrophages may play a more complex and significant role in the disease process than has been previously reported, with activated macrophages accumulating in infected hyperplastic crypts. Sci STKE . 2003 Jun 24;2003(188):re10. Voltage-gated K channels; Armstrong CM; Ion channels and the electrical properties they confer on cells are involved in every human characteristic that distinguishes us from the stones in a field . Every perception, thought, movement, and heartbeat depends on electrical signals generated by the activity of ion channels . Early views of the relationship between channel structure and function have undergone substantial modification following the cloning of various ion channels and the determination of the structure of a simple bacterial K channel, the KcsA channel . This review focuses on the relationship between the structure and function of voltage-dependent K channels, covering the molecular bases of channel selectivity, conduction, and gating . The evolution of ion channels in bacteria is discussed, as well as the basis of channel selectivity and conduction in the KcsA channel . More complex channels have evolved molecular "gatekeepers," allowing them to respond to appropriate stimuli by opening, closing, and inactivating. Med Vet Entomol, 2003 Jun, 17(2), 115 - 32 Dipteran predators of Simuliid blackflies: a worldwide review; Werner D et al.; Haematophagous female blackflies (Diptera: Simuliidae) are serious biting pests and obligate vectors of vertebrate pathogens, namely filarial Dirofilaria, Mansonella, Onchocerca and protozoal Leucocytozoon . Immature stages of Simuliidae inhabit lotic waterways, the sessile larvae filter-feeding and often forming a large proportion of the benthic biomass, usually aggregated in well-oxygenated sections of streams, rivers, waterfalls and spillways . Simuliid control practices depend on larvicidal chemicals, biological products (bacteria, nematodes) and environmental modification . The potential use of predators for biological control of Simuliidae has not been exploited . Predators of Simuliidae include examples of at least 12 families of Diptera and other predaceous arthropods (Crustacea and insects: Coleoptera, Odonata, Plecoptera, Trichoptera), invertebrates (notably Turbellaria), as well as browsing fish . Diptera impacting upon simuliid populations comprise mainly Chironomidae, Empididae and Muscidae, although several other families (Asilidae, Dolichopodidae, Phoridae, Drosophilidae, Scathophagidae) play a significant role as predators . Details of predator and prey species and life stages are presented, by zoogeographical region, including the prevalence of cannibalism among Simuliidae. Mol Microbiol, 2003 Jul, 49(1), 219 - 34 A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system; Rohde M et al.; Type I strains of Helicobacter pylori (Hp) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island (cag-PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin-8 secretion . Translocated CagA is activated by tyrosine phosphorylation involving Src-family kinases . The mechanism and structural basis for type IV protein secretion is not well understood . We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS . The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria . It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10-homologous protein . HP0527 is also clustered in the outer membrane . The VirB7-homologous protein HP0532 is found at the base of this organelle . These observations demonstrate for the first time by microscopic techniques a complex T4SS-associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems. Evol Dev, 2003 Jul-Aug, 5(4), 379 - 85 Rickettsia-like mitochondrial motility in Drosophila spermiogenesis; Bazinet C et al.; Although it is generally accepted that mitochondria and chloroplasts are descended in evolution from bacteria, the potential contributions of their endosymbiont ancestors to specialized cellular pathways in development remain largely unexplored . Here we show that a motile behavior of mitochondria in Drosophila spermiogenesis is strikingly similar to the actin-based "comet tail" motility of several bacteria . A combination of electron and fluorescence microscopy demonstrates major reorganization and movement of mitochondria ahead of, and in close association with, dense conical arrays of actin filaments in the sperm individualization complex, which mediates the resolution of male germline syncytia into separate gametes . Because of several other parallels between the movement of the individualization complex and the motility behavior of some rickettsiae, the bacterial family from which mitochondria are most likely descended, this motility phenomenon is a strong candidate for a true vestige of endosymbiont behavior in contemporary mitochondria . The potential conservation of an ancient endosymbiont motility mechanism within a highly conserved feature of gametogenesis, the resolution of germline syncytia, may indicate a formative role for the endosymbiotic ancestor of mitochondria in the evolution of this developmental pathway. Artif Organs, 2003 Jul, 27(7), 623 - 30 Liposomal entrapment of cefoxitin to improve cellular viability and function in human saphenous veins; Park JC et al.; Liposomal cefoxitin was prepared and applied to the pretreatment of human saphenous vein (HSV) for implantation . The possible use of liposomal cefoxitin to improve cellular viability and function and to maintain its potential sterilization effect was investigated . Entrapment efficiency and size distribution of liposomal cefoxitin were 75.7% and 652 +/- 75.7 nm, respectively . The weight ratio between cefoxitin and liposome was calculated at 1 : 40.6 . When cefoxitin was entrapped with liposome, the released amount of cefoxitin was not affected by temperature conditions (37 degrees C, 25 degrees C, and 4 degrees C) . The amount of free cefoxitin present in HSV reached 59% at 0.5 h and gradually decreased with time, while liposomal cefoxitin showed a maximum amount (63%) at 1.5 h, indicating that liposomal cefoxitin seemed to control the initial amount of cefoxitin present in HSV . Liposomal cefoxitin showed better viabilities of whole cells and endothelial cells dissociated from HSV than free cefoxitin and remarkably superior function of endothelial cells, as determined by Griffonia simplicifolia agglutinins-fluorescein isothiocyanate/propidium iodide double-staining methods combined with flow cytometry and endothelial nitric oxide synthase assay, respectively . In terms of sterilization effect, there was no significant difference between liposomal cefoxitin and free cefoxitin . These results suggest that liposomal entrapment of cefoxitin could improve cellular viability and functions and maintain the original sterilization effect. Clin Exp Immunol, 2003 Jul, 133(1), 30 - 7 A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes; Lopez B et al.; In the last decade, an unprecedented genetic diversity has been disclosed among Mycobacterium tuberculosis strains found worldwide . However, well-conserved genotypes seem to prevail in areas with high incidence of tuberculosis . As this may be related to selective advantages, such as advanced mechanisms to circumvent {M . bovis Bacille Calmette-Guerin (BCG)-induced} host defence mechanisms, we investigated the influence of strain diversity on the course of experimental disease . Twelve M . tuberculosis strains, representing four major genotype families found worldwide today, and the laboratory strain H37Rv were each used to infect BALB/c mice by direct intratracheal injection . Compared with H37Rv, infections with Beijng strains were characterized by extensive pneumonia, early but ephemeral tumour necrosis factor-alpha (TNF-alpha) and inducible isoform of nitric oxide synthetase (iNOS) expression, and significantly higher earlier mortality . Conversely, Canetti strains induced limited pneumonia, sustained TNF-alpha and iNOS expression in lungs, and almost 100% survival . Strains of the Somali and the Haarlem genotype families displayed less homogeneous, intermediate rates of survival . Previous BCG vaccination protected less effectively against infection with Beijing strains than against the H37Rv strain . In conclusion, genetically different M . tuberculosis strains evoked markedly different immunopathological events . Bacteria with the Beijing genotype, highly prevalent in Asia and the former USSR, elicited a non-protective immune response in mice and were the most virulent . Future immunological research, particularly on candidate vaccines, should include a broad spectrum of M . tuberculosis genotypes rather than a few laboratory strains. Dig Dis Sci, 2003 Jun, 48(6), 1165 - 70 Obstructive jaundice alters LFA-1alpha expression in rat small intestine; Sheen-Chen SM et al.; Translocation of bacteria and endotoxtin has long been documented in obstructive jaundice, and altered intestinal barrier function is considered to be one of the important mechanisms for this phenomenon . The regulation of gastrointestinal mucosal response to injury is thus of important clinical as well as biological relevance . Integrins play a critical role in enterocyte migration, which is essential to mucosal healing . This study is designed to evaluate the integrins status in obstructive jaundice . Male Sprague-Dawley rats (N = 37) were randomized to three groups . Group 1 (N = 12) underwent common bile duct ligation (CBDL), group 2 (N = 12) underwent common bile duct ligation with oral glutamine administration (CBDL + G), and group 3 (N = 13) underwent a sham operation (sham control) . After seven days, segments of proximal jejunum and distal ileum were harvested, and cell surface immunohistochemical expression of LFA-1alpha and VLA-6 were evaluated and recorded . The staining intensities were graded on a scale of 0-4 . Comparisons among the three groups were performed . There was no significant difference in VLA-6 staining on small intestine among the three groups (P > 0.05) . There was also no significant difference in LFA-1alpha staining the on jejunum between group 1 (CBDL) and group 3 (sham control) (P > 0.05) . However, the LFA-1alpha staining on the ileum in group 1 (CBDL) significantly decreased when compared with group 3 (sham control) (P = 0.008) . With oral glutamine administration (0.2 g/kg body weight, once daily), LFA-1alpha staining on the ileum was significantly restored in group 2 (CBDL + G) . In conclusion, obstructive jaundice for one week down-regulates LFA-1alpha expression on rat ileum . With oral glutamine administration, such down-regulation of LFA-1alpha expression on rat ileum can be restored . Such a phenomenon is intriguing and deserves further evaluation and elucidation. Crit Rev Food Sci Nutr, 2003, 43(3), 245 - 64 A review on biological control and metabolism of aflatoxin; Mishra HN et al.; The series of events that led to the discovery of aflatoxin as a potent carcinogen, its biosynthesis, mechanism of action, structure-function relationship provide interesting insight into the economical and technological factors involved in the development of an effective control measure for the toxin . Scientists all over the world are making continuous efforts to explore a generalized process of detoxification, which can bring down the toxin content in heterogenous commodities to a threshold level . In this article biological control methods with special emphasis on in vivo and in vitro enzymatic detoxification of aflatoxin have been reviewed . Future areas of research involving large-scale enzymatic detoxification and modified atmosphere storage are also discussed. Oncogene, 2003 Jun 26, 22(26), 3992 - 7 Role of nuclear and cytoplasmic localization in the tumour-suppressor activity of the von Hippel-Lindau protein; Lewis MD et al.; Mutations in the von Hippel-Lindau (VHL) tumour-suppressor gene result in several forms of cancer . In the present study, we investigated the role of VHL subcellular localization in its antitumour properties . We generated renal cell carcinoma (RCC) lines stably expressing either exclusively nuclear (RCC/NLS-VHL), cytoplasmic (RCC/NES-VHL) or nucleo-cytoplasmic shuttling (RCC/DeltaNES-VHL or RCC/VHL) forms of VHL and investigated several parameters linked to tumorigenesis and known to be dysregulated in VHL disease . Remarkably, although the expression of wild-type VHL is largely cytoplasmic, all of the antitumour properties of VHL tested could be reconstituted by expressing exclusively nuclear VHL. Biochem Biophys Res Commun, 2003 Jul 11, 306(4), 872 - 9 Light plays a key role in the modulation of heat shock response in the cyanobacterium Synechocystis sp PCC 6803; Asadulghani et al.; The heat shock response is generally characterized by an immediate, intense, and transient activation of gene expression, resulting in the elevated synthesis of heat shock proteins . We found that light modulates these characteristics of the heat shock response in cyanobacteria . Light accelerated the heat induction of htpG, groESL1, groEL2, and hspA, in Synechocystis sp . PCC 6803 . In the dark, heat shock response of all the heat shock genes except hspA was not as intense as in the light and no transient peak was detected within 3h after heat shock over the time course of the hspA and groESL1 mRNA accumulation . There was an apparent relationship between the enhancement of the heat shock gene transcription in the light and the level of reduced plastoquinone in the photosynthetic electron transport system . Light affected the transcription, but not the stability of the mRNA of heat shock genes, although the stability was quite different, depending on the heat shock gene . Light also enhanced both the accumulation of GroEL under heat stress and the acquired thermo-tolerance. Zhonghua Yi Xue Za Zhi, 2003 Mar 10, 83(5), 391 - 4 {Efficacy and safety of cefditoren pivoxil in treatment of respiratory infections, a clinical study}; Li JT et al.; OBJECTIVE: To evaluate the efficacy and safety of cefditoren pivoxil in treatment of respiratory infections . METHODS: 199 cases of respiratory infection confirmed by etiological and clinical examinations were treated with cefditoren pivoxil tablets taken orally . Therapeutic evaluation was conducted among 196 cases and safety evaluation was conducted among 199 cases . RESULTS: The total effective rate was 94.9%, and the causative bacteria -elimination rate was 96.7% . Clinical adverse events, including moderate diarrhea, mild nausea and vomiting, and stomach discomfort, were seen in 9 cases with an incidence rate of 4.5% . Laboratory adverse events, including increase of with an incidence rate of 3.5% . CONCLUSION: Cefditoren pivoxil is effective and safe in treatment of mild and moderate respiratory infections . The resistance rate to cefditoren pivoxil of pathogens of respiratory infections and the efficacy of cefditoren pivoxil show no difference from those tested 7 years ago. Biochemistry, 2003 Jul 1, 42(25), 7701 - 8 A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure; Dunham CM et al.; To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation . In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened . Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL . In contrast, the level of binding of oxygen was dramatically lower (K(d) approximately 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively . Binding of cyanide followed the same pattern as binding of oxygen . The catalytic activity was 3-4-fold higher in the "on-state" unliganded forms of R220A BjFixL than in the corresponding BjFixL species . Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved . Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL . We conclude that the G(beta)-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form . This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands . These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch. J Vet Sci, 2003 Apr, 4(1), 41 - 9 Mac-1-mediated uptake and killing of Bordetella bronchiseptica by porcine alveolar macrophages; Lee JK et al.; The role of Mac-1 as a receptor for Bordetella bronchiseptica infection of alveolar macrophages (AMphi) was examined using 6 strains (2 ATCC and 4 pathogenic field isolates) to assess B . bronchiseptica binding, uptake and replication in primary porcine AMphi . All B . bronchiseptica strains were rapidly killed by porcine serum in a dose- and time-dependent manner . However, heat-inactivated porcine serum (HIS) did not demonstrate any bacterial-killing activity, suggesting that complement may have a direct killing activity . All field isolates were more resistant to direct complement-mediated B . bronchiseptica killing . The uptake of B . bronchiseptica into AMphi was inhibited approximately 50% by anti-Mac-1 monoclonal antibodies in the medium . However, B . bronchiseptica phagocytosed in the presence of serum or HIS was not altered by anti-Mac-1 antibodies although more bacteria were internalized by addition of serum or HIS . These data suggest that Mac-1 is a target for direct uptake of B . bronchiseptica via opsonin-independent binding . The phagocytosed B . bronchiseptica, either via direct or serum-mediated binding, were efficiently killed by AMphi within 10 hr postinfection . This demonstrates that Mac-1-mediated B . bronchiseptica uptake is a bacterial killing pathway not leading to productive infections in AMphi. J Biol Chem, 2003 Sep 12, 278(37), 35558 - 63 Epub 2003 Jun 20. Loop swapping in an antisense RNA/target RNA pair changes directionality of helix progression; Slagter-Jager JG et al.; The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps . It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems . This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B' . Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression . Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics . Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended . In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn. Infect Immun, 2003 Jul, 71(7), 4137 - 43 Bordetella bronchiseptica persists in the nasal cavities of mice and triggers early delivery of dendritic cells in the lymph nodes draining the lower and upper respiratory tract; Gueirard P et al.; Early after the intranasal instillation of Bordetella bronchiseptica into mice, not only are mature dendritic leukocytes recovered from lung parenchyma and bronchoalveolar lavage fluid but their numbers are also increased in the mediastinal lymph nodes and the nasal mucosa-associated lymphoid tissue . Later during the infectious process, the bacteria persist mainly in the nasal cavity. Infect Immun, 2003 Jul, 71(7), 3837 - 43 Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori; Voland P et al.; Outer membrane proteins (OMPs) are incorporated into the outer plasma membrane of Helicobacter pylori and are important for, e.g., ion transport, adherence, structural and osmotic stability, and bacterial virulence but may also be antigenic due to their surface exposure . Previous proteome-based approaches with H . pylori lysates determined a strong serological reaction towards two H . pylori OMPs, HpaA (TIGR HP0797) and Omp18 (TIGR HP1125) . PCR was used to detect DNA encoding the two proteins, and a positive signal was found in all H . pylori strains tested . Proteins were cloned and expressed in the human kidney cell line HK293 with the QiaExpressionist system with a C-terminal His tag . Only sera from infected persons showed a positive reaction with the recombinant proteins . Recombinant HpaA (rHpaA) and rOmp18 were incubated with human peripheral blood mononuclear cells and induced secretion of interleukin-12 (IL-12) and IL-10 from these cells . To determine the effect on antigen-presenting cells, human blood monocytic and dendritic cells (DCs) were isolated by magnetic cell separation . rOmp18 and rHpaA strongly stimulated major histocompatibility class II and CD83 expression 7- to 10-fold on isolated DCs . rHpaA and rOmp18 failed to stimulate IL-8 secretion from monocytes but increased secretion of IL-12 and IL-10 from DCs significantly . In summary, HpaA and Omp18 are recognized by human dendritic cells and induce their maturation as well as antigen presentation . HpaA and Omp18 of H . pylori thereby appear to have a specific antigenic potential in humans. Dev Comp Immunol, 2003 Oct, 27(9), 813 - 22 Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss; Zou J et al.; Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response . Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss . In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro . Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1beta, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11 . The stimulatory doses of both rTNFs were >or=10 ng/ml . Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner . Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities. J Mol Biol, 2003 Jun 27, 330(1), 113 - 28 The active conformation of glutamate synthase and its binding to ferredoxin; van den Heuvel RH et al.; Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate . The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases . The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain . Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form . The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis . Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex . These structures represent the enzyme in the active conformation . By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites . X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd) . Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd . A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis. Immunity, 2003 Jun, 18(6), 813 - 23 Legionella reveal dendritic cell functions that facilitate selection of antigens for MHC class II presentation; Neild AL et al.; To understand how adaptive immune responses are generated against bacteria that avoid being delivered to lysosomes, interactions between professional antigen-presenting cells (APCs) and the intracellular pathogen Legionella pneumophila were examined . In contrast to murine bone marrow-derived macrophages (BMMs), we show that dendritic cells (DCs) restrict the growth of intracellular Legionella . Similar to what has been reported in BMMs, phagosomes containing Legionella matured into endoplasmic reticulum (ER)-derived organelles after DC internalization . Biogenesis of an ER-derived vacuole did not effectively sequester Legionella antigens from presentation on MHC class II molecules (MHC II) . It was determined that proteins synthesized after Legionella had established residence in an ER-derived vacuole were presented by infected APCs . These data indicate that the ability of DCs to restrict intracellular growth of Legionella could be an important property that facilitates priming of protective T cell-mediated immune responses to vacuolar pathogens. Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8828 - 33 Epub 2003 Jun 19. High-frequency gene transfer from the chloroplast genome to the nucleus; Stegemann S et al.; Eukaryotic cells arose through endosymbiotic uptake of free-living bacteria followed by massive gene transfer from the genome of the endosymbiont to the host nuclear genome . Because this gene transfer took place over a time scale of hundreds of millions of years, direct observation and analysis of primary transfer events has remained difficult . Hence, very little is known about the evolutionary frequency of gene transfer events, the size of transferred genome fragments, the molecular mechanisms of the transfer process, or the environmental conditions favoring its occurrence . We describe here a genetic system based on transgenic chloroplasts carrying a nuclear selectable marker gene that allows the efficient selection of plants with a nuclear genome that carries pieces transferred from the chloroplast genome . We can select such gene transfer events from a surprisingly small population of plant cells, indicating that the escape of genetic material from the chloroplast to the nuclear genome occurs much more frequently than generally believed and thus may contribute significantly to intraspecific and intraorganismic genetic variation. Blood, 2003 Dec 15, 102(13), 4329 - 35 Epub 2003 Jun 19. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates; Horn PA et al.; The nonobese diabetic/severe combined immune-deficient (NOD/SCID) mouse xenotransplantation assay is the most commonly used surrogate assay for the study of human candidate stem cells . In contrast to large animal and human studies, however, it is limited by the short life span of the recipients, the limited proliferative demand placed on the transplanted cells, and the inability to support differentiation into all hematopoietic lineages . In the present study, we directly compared hematopoietic repopulation in NOD/SCID mice with autologous reconstitution in the baboon, a well-established preclinical large animal model for stem cell transplantation . Baboon CD34-enriched marrow cells were retrovirally marked and infused into the irradiated baboon and the NOD/SCID mice . Although the percentage of gene-marked cells was high and remained stable in NOD/SCID mice up to 12 weeks and in those that underwent secondary transplantation, we observed a considerable decline and overall a significantly (10-fold) lower percentage of gene-marked cells in the baboons . In addition, clonal integration site analysis revealed common proviral vector integrants in NOD/SCID repopulating cells and in the baboon at 6 weeks but not at 6 months after transplantation . These results suggest that distinct hematopoietic stem/progenitor cells are responsible for hematopoietic reconstitution in NOD/SCID mice compared with nonhuman primates. Philos Transact A Math Phys Eng Sci, 2003 Jun 15, 361(1807), 1187 - 203 Self-organization: making complex infectious viral particles from purified precursors; Bamford DH; Viruses have served as excellent model systems in which to study biological self-organization . Purified virion structural constituents have been shown to self-assemble into particles that can initiate a productive infection in the host cell resulting in the release of progeny virions . Accumulating information on virus structures and assembly principles has revealed unexpected similarities between viruses that infect hosts as diverse as bacteria and humans, suggesting that these viruses had an early common ancestor . I will describe, in more detail, the assembly pathway of a complex double-stranded RNA bacterial virus . In this system, infectious viral particles are produced starting from purified protein and nucleic acid constituents through an elaborate self-assembly, RNA-packaging and synthesis pathway. Filaria J . 2003 May 2;2(1):9. Evidence against Wolbachia symbiosis in Loa loa; McGarry HF et al.; BACKGROUND: The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection . Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease . Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa . Here we use molecular and immunohistological techniques to confirm this finding . METHODS: We have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA {rDNA}, ftsZ and Wolbachia surface protein {WSP}) on samples of L . loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L . loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts . Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3 . The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M . streptocerca and Wuchereria bancrofti . Immunohistology was carried out by light and electron microscopy on L . loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP . Samples from nematodes known to be infected with Wolbachia (O . volvulus, O . ochengi, Litomosoides sigmodontis and B . malayi) were used as positive controls and A . viteae as a negative control . RESULTS: Single PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L . loa . Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions . The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers . Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis . Immunohistology of L . loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis . CONCLUSION: DNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L . loa. J Periodontol, 2003 May, 74(5), 590 - 6 Periodontal treatment with an Er:YAG laser or scaling and root planing . A 2-year follow-up split-mouth study; Schwarz F et al.; BACKGROUND: Non-surgical periodontal treatment with an Er:YAG laser has been shown to result in significant clinical attachment level gain; however, clinical results have not been established on a long-term basis following Er:YAG laser treatment . Therefore, the aim of the present study was to present the 2-year results following non-surgical periodontal treatment with an Er:YAG laser or scaling and root planing . METHODS: Twenty patients with moderate to advanced periodontal destruction were treated under local anesthesia, and the quadrants were randomly allocated in a split-mouth design to either 1) Er:YAG laser (ERL) using an energy level of 160 mJ/pulse and 10 Hz, or 2) scaling and root planing (SRP) using hand instrumen