Microorganisms

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 instruments . The following clinical parameters were evaluated at baseline and at 1 and 2 years after treatment: plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), gingival recession (GR), and clinical attachment level (CAL) . Subgingival plaque samples were taken at each appointment and analyzed using dark-field microscopy for the presence of cocci, non-motile rods, motile rods, and spirochetes . The primary outcome variable was CAL . No statistically significant differences between the groups were found at baseline . Power analysis to determine superiority of ERL treatment showed that the available sample size would yield 99% power to detect a 1 mm difference . RESULTS: The sites treated with ERL demonstrated mean CAL change from 6.3 +/- 1.1 mm to 4.5 +/- 0.4 mm (P < 0.001) and to 4.9 +/- 0.4 mm (P < 0.001) at 1 and 2 years, respectively . No statistically significant differences were found between the CAL mean at 1 and 2 years postoperatively . The sites treated with SRP showed a mean CAL change from 6.5 +/- 1.0 mm to 5.6 +/- 0.4 mm (P < 0.001) and to 5.8 +/- 0.4 mm (P < 0.001) at 1 and 2 years, respectively . The CAL change between 1 and 2 years did not present statistically significant differences . Both groups showed a significant increase of cocci and non-motile rods and a decrease in the amount of spirochetes . However, at the 1- and 2-year examination, the statistical analysis showed a significant difference for the CAL (P < 0.001, respectively) between the 2 treatment groups . CONCLUSION: It was concluded that the CAL gain obtained following non-surgical periodontal treatment with ERL or SRP can be maintained over a 2-year period.

Mol Pharmacol, 2003 Jul, 64(1), 11 - 20
Spatial regulation of Galphai protein signaling in clathrin-coated membrane microdomains containing GAIP; Elenko E et al.; Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to Galpha subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established . Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous Galpha-interacting protein (GAIP) (RGS19) binds to Galphai3-YFP and its fate after activation of delta-opioid receptor (DOR) . In the absence of agonist, GAIP is spatially segregated from Galphai3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Galphai3-YPF and DOR are located in non-clathrin-coated microdomains of the PM . Upon addition of agonist, Galphai3 partially colocalizes with GAIP in CCPs at the PM . When endocytosis is blocked by expression of a dynamin mutant {dyn(K44A)}, there is a striking overlap in the distribution of DOR and Galphai3-YFP with GAIP in CCPs . Moreover, Galphai3-YFP and GAIP form a coprecipitable complex . Our results support a model whereby, after agonist addition, DOR and Galphai3 move together into CCPs where Galphai3 and GAIP meet and turn off G protein signaling . Subsequently, Galphai3 returns to non-clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles . This constitutes a novel mechanism for regulation of Galpha signaling through spatial segregation of a GAP in clathrin-coated pits.

Eur Urol, 2003 Jul, 44(1), 1 - 7
Sexually transmitted diseases (STDs)--a synoptic overview for urologists; Schneede P et al.; The classical bacteria that cause venereal diseases, e.g . gonorrhea, syphilis, chancroid and inguinal granuloma only account for a small proportion of all known STDs today . Other bacteria and viruses as well as yeasts, protozoa and epizoa must also be regarded as causative organisms of STD . Taken together, all sexually transmitted infections (STI) comprise more than 30 relevant STD pathogens . However, not all pathogens that can be sexually transmitted manifest diseases in the genitals and not all infections of the genitals are exclusively sexually transmitted . Concise information and tables summarising the diagnostic and therapeutic management of STDs in the field of Urology allow a synoptic overview and are in agreement with recent international guidelines of other specialties . Special considerations (i.e . HIV infection, pregnancy, infants, allergy) and recommended regimens may be looked up here.

Int J Parasitol, 2003 Jul, 33(7), 691 - 702
Molecular characterisation of EmTFP250: a novel member of the TRAP protein family in Eimeria maxima; Witcombe DM et al.; We have previously described a high molecular mass, asexual stage antigen from Eimeria maxima (EmTFP250), implicated as a target of maternal antibodies produced by breeding hens infected with this protozoan parasite . Following partial purification of the protein by ion exchange chromatography, N-terminal and internal peptide sequences were generated and used in the design of degenerate PCR primers . Using a rapid amplification of cDNA ends PCR-based strategy, the cDNA encoding EmTFP250 has been cloned and sequenced . Translation predicts a mature polypeptide with a molecular mass of 246kDa and an isoelectric point of 4.2 . Analysis of the amino acid sequence has revealed a novel member of the TRAP (thrombospondin-related anonymous protein) family, containing 16 thrombospondin type-1 repeats and 31 epidermal growth factor-like calcium binding domains . EmTFP250 also contains two low complex, hydrophilic regions rich in glutamic acid and glycine residues, and a transmembrane domain/cytosolic tail associated with parasite gliding motility that is highly conserved within apicomplexan microneme proteins . The protein has 61% identity (71% similarity) with EtMIC4, a 218kDa microneme protein of Eimeria tenella also rich in epidermal growth factor-like and thrombospondin type-1 domains . Using Southern blotting, the gene encoding EmTFP250 has been determined to be present as a single copy within the genome, and reverse transcriptase-PCR has shown that expression is confined to the asexual stages of development . By employing a PCR-based method, a region of the E . maxima Houghton strain EmTFP250 gene was found conserved in Australian isolates of several (at least four) Eimeria species that parasitise chickens . The characterisation of EmTFP250 adds to the expanding apicomplexan TRAP family and suggests a functional significance for the protein.

J Biol Chem, 2003 Sep 26, 278(39), 37511 - 9 Epub 2003 Jun 17.
Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure, function, and stability; Campanini B et al.; O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism . The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain . Only Trp161 is highly conserved in eucaryotes and bacteria . The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains . The enzyme undergoes an open to closed conformational transition upon substrate binding . The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques . The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction . The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface . The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.

J Biol Chem, 2003 Aug 29, 278(35), 32962 - 8 Epub 2003 Jun 17.
Interferon-gamma augments CARD4/NOD1 gene and protein expression through interferon regulatory factor-1 in intestinal epithelial cells; Hisamatsu T et al.; Although intestinal epithelial cells appear to be functionally hyporesponsive to normal intestinal flora, human intestinal epithelial cells can respond to enteroinvasive bacteria and induce an inflammatory response . This initial inflammatory response leads to the recruitment of polymorphonuclear leukocytes to the affected site in vitro and in vivo . CARD4/NOD1 is a potential cytosolic receptor for peptidoglycan in mammalian cells that resembles pathogen-resistant proteins of plants . In this context, CARD4/NOD1 is a candidate for a recognition protein of intracellular bacteria or peptidoglycan in intestinal epithelial cells . In this study, we demonstrate that CARD4/NOD1 is constitutively expressed in intestinal epithelial cell lines and isolated primary intestinal epithelial cells . Interferon-gamma (IFN gamma), which is a potent pro-inflammatory cytokine in intestinal mucosal inflammation, activates CARD4/NOD1 mRNA transcription in a time- and dose-dependent manner and results in augmentation of CARD4/NOD1 protein in SW480 cells . Promoter analysis of CARD4/NOD1 indicates that interferon regulatory factor-1 (IRF-1) binding motif (-791 to -782) is essential for the effect of IFN gamma . Nuclear extracts from SW480 cells treated with IFN gamma show specific binding of oligonucleotides corresponding to this IRF-1-binding motif, which was supershifted by anti-IRF-1 antibody in electrophoretic mobility shift assay . Overexpression of IRF-1 protein activates the CARD4/NOD1 promoter but not the deletion mutant of the IRF-1-binding site in a co-transfection assay of IRF-1 expression plasmid with CARD4/NOD1 promoter . These studies suggest that the Th1 cytokine, IFN gamma, activates CARD4/NOD1 transcription and regulate innate immune mechanisms in the condition of intestinal mucosal inflammation.

Zhonghua Yi Xue Za Zhi, 2003 Feb 10, 83(3), 232 - 6
{Effect of exogenous epitopes of helper T lymphocyte on humoral immunity of HBV S gene DNA immunity}; Peng XM et al.; OBJECTIVE: To study the effect of exogenous epitope of helper T lymphocyte (HTL) on humoral immunity of HBV S gene DNA immunity . METHODS: Two universal HTL epitopes, amino acid residue (aa) 830-843 of the tetanus toxoid (TTE) and artificial epitope (PADRE), and 3 unique epitopes, aa1-20 of tubercle bacteria hot shock protein 65 (TBE), aa54-65 of rubella protein E2-4 (ME) and aa35-48 of trachoma hot shock protein 60 (CE) were chosen . Eukaryotic expression vectors were constructed by inserting single or multiple exogenous epitopes in HBV S gene just after the initial code of translation . BALB/c mice were inoculated with 100 micro g of recombinant DNA per mouse, and given boost inoculation for 3 times with 3-week interval . Mouse blood were collected one month just after the third boost inoculation . Anti-HBs was detected using Abbott test kits . RESULTS: HBV S eukaryotic gene expression vectors, pHB and 6 exogenous HTL epitope HBV S gene vectors, pHB-TBE, pHB-PADRE, pHB-TTE, pHB-MTE2, pHB-MTE3 and pHB-MTE5 were constructed successfully with anti-HBs level (IU/L) of 10 +/- 5, 5 +/- 5, 49 +/- 7, 29 +/- 6, 16 +/- 8, 23 +/- 7 and 28 +/- 8 respectively . Among 3 single epitopes, TTE and PADRE had obviously effect on promoting the anti-HBs response of HBV S gene, while TBE had no promoting effect . All of the 3 multiple epitopes were shown the effect of immune promoting . CONCLUSION: Some exogenous HTL epitopes had obviously effect on promoting the anti-HBs response of HBV S gene . Multiple epitopes also had humoral immunity promoting effect, but there was no synergic effect among their own HTL epitopes . PADRE might be an important candidate for new efficient HB vaccine . The multiple epitope cluster consisted form 5 exogenous epitopes might be an important candidate for the reinoculating HB vaccine or therapy HB vaccine.

Protist, 2003 Apr, 154(1), 91 - 8
Mixotrophy of a photosynthetic flagellate viewed from an optimal foraging perspective; Stibor H et al.; Mixotrophy, a combination of phototrophic and phagotrophic nutrition, has been found in several classes of phytoplankton (Booras et al . 1988, Jones 2000) and appears to be a successful evolutionary strategy . Heterotrophic nutrition of phytoplankton has been suggested to be an important source of mineral nutrients (Nygaard and Tobiesen 1993) . Potentially limiting mineral nutrients, particularly phosphorus (P), are often several orders of magnitude more concentrated in the biomass of food organisms of mixotrophs (e.g . in bacteria) than in the dissolved phase (Vadstein 2000) . We used radioactive tracer experiments to show that the simultaneous uptake of P from dissolved inorganic and particular P sources by the marine phytoflagellate Chrysochromulina polylepis followed basic predictions of optimal foraging theory (Stephens and Krebs 1986) . Chrysochromulina takes up its P rather unselectively from both bacterial P and dissolved P sources at low dissolved P concentrations, while it becomes more selective at higher dissolved inorganic P (DIP) concentrations . The onset of mixotrophic processes was dependent on DIP concentrations . These findings support the view of mixotrophy as a strategy of nutrient uptake in nutrient poor (oligotrophic) pelagic environments (Nygaard and Tobiesen 1993) and show that ideas of optimal foraging can be applied to unicellular organisms.

Protist, 2003 Apr, 154(1), 43 - 55
Making a robust biomolecular time scale for phylogenetic studies; Foster KW; The further evolution of informational molecular sequences should depend on the number of viable alternatives possible for the sequences as set by selection, the unrepaired mutation rate, and time . Most biomolecular clocks are based on Kimura's nearly neutral mutation random-drift hypothesis . This clock assumes that informational sequences are in equilibrium, i.e., the nucleotides mutate at a uniform rate and the number of nucleotides unconstrained by selection remains constant . Correcting for deviations from these assumptions should produce a more accurate clock . Informational molecules probably formed from polynucleotides having some other function such as nitrogen or nucleotide storage, thus being initially functionally unselected . At any time the rate of development of functionality in a protein may be expected to be proportional to the number of viable alternatives of sequence in its potentially interacting regions . Assuming the rate of unrepaired mutations is constant, these clocks should exponentially slow as they evolve, each with a different rate toward individual equilibria . Also if the degree of selection changes, its clock rate should change . For a more precise clock two approaches are suggested to estimate these time dependent changes in evolutionary rate . An improved clock could improve estimation of phylogeny and put a time scale on that phylogeny.

Clin Chem Lab Med, 2003 May, 41(5), 640 - 5
Early detection of peritonitis in continuous ambulatory peritoneal dialysis patients by cytokine measurements; Oymak Y et al.; Continuous ambulatory peritoneal dialysis (CAPD) carries a risk of peritonitis which is accompanied by mild symptomatology . Culture of effluent has yielded organism in 50% of cases . Peritoneal phagocytes produce tumor necrosis factor-alpha and interleukin (IL)-1 in response to contact with bacteria, initiating an inflammatory cascade which leads to IL-6 and IL-8 secretion . Additonally, neutrophils undergo an increase in oxidative metabolism . We have evaluated the diagnostic accuracy of effluent measurements of TNF-alpha, IL-6, IL-8, and oxidative metabolism markers in these patients . Dialysate fluids (n = 65) were collected from non-infected patients and those presenting with acute peritonitis . Positive culture proved the diagnosis . Oxidative markers and nitric oxide were measured by chemiluminescence . Cytokines were measured by solid phase chemiluminescent immunometric assay (Immulite, DPC, USA) . Receiver operating characteristic (ROC) curves were used to assess the diagnostic accuracy and the areas under curves were calculated for comparison . All effluent cytokines and oxidative markers were significantly higher in patients with peritonitis when compared to those without (p < 0.05) . Significant correlations were evident between IL-6 and IL-8, lucigenin chemiluminescence and luminol chemiluminescence, lucigenin chemiluminescence and IL-6 or IL-8, and luminol chemiluminescence and IL-6 or IL-8 . ROC curves showed that the ability of IL-6, IL-8, lucigenin chemiluminescence, and luminol chemiluminescence to differentiate CAPD patients with peritonitis from non-infected cases exceeds that of polymorphonuclear leukocyte count.

Rev Physiol Biochem Pharmacol, 2003, 150, 1 - 35 Epub 2003 Jun 17.
Structure-function relationship in P-type ATPases--a biophysical approach; Apell HJ; P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes . In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane . P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions . So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase . However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps . The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure-function relationship.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7539 - 44 Epub 2003 Jun 16.
An unusual type IB topoisomerase from African trypanosomes; Bodley AL et al.; African trypanosomes are ancient eukaryotes that cause lethal disease in humans and cattle . Available drugs are inadequate and the need for new therapeutic targets is great . Trypanosoma brucei and related pathogens differ strikingly from higher eukaryotes in many aspects of nucleic acid structure and metabolism . We find yet another example of this in their unusual DNA topoisomerase IB . Type IB topoisomerases relieve the supercoils that accumulate during DNA and RNA synthesis, and are of considerable importance as the target for antitumor camptothecins . Dozens of type IB topoisomerases sequenced from eukaryotes, bacteria, and pox viruses are all encoded by a single gene that predictably contains a highly conserved DNA binding domain and C-terminal catalytic domain, linked by a nonconserved hydrophilic region . We find that topoisomerase IB in T . brucei is encoded by two genes: one for the DNA-binding domain and a second for the C-terminal catalytic domain . In keeping with this, highly purified fractions of native T . brucei topoisomerase IB catalytic activity contain two proteins, of 90 and 36 kDa . The native enzyme is conventional in its Mg2+-independence, ability to relax positive and negative supercoils, and inhibition by camptothecin . Camptothecin promotes the formation of a covalent complex between 32P-labeled substrate DNA and the small subunit . This unusual structural organization may provide a missing link in the evolution of type IB enzymes, which are thought to have arisen over time from the fusion of two independent domains . It also provides another basis for the design of selectively toxic drug candidates.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7824 - 9 Epub 2003 Jun 16.
Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis; Su T et al.; PKC-interacting protein (PKCI), also designated histidine triad nucleotide-binding protein 1, belongs to the histidine triad (HIT) family of proteins . Its structure is highly conserved from bacteria to humans and shares homology with the tumor-suppressor gene fragile histidine triad (FHIT) . Although it was originally thought to inhibit PKC, its actual physiologic function is not known . Therefore, we used the technique of homologous recombination to generate homozygous deleted PKCI-/- mice . These mice display normal fetal and adult development . However, when mouse embryo fibroblasts were established from 13.5-day embryos and serially passaged the PKCI-/- cells displayed an increase in growth rate and underwent spontaneous immortalization, whereas the PKCI+/+ cells senesced and ceased growing . Furthermore, the PKCI-/- mouse embryo fibroblasts displayed increased resistance to cytotoxicity by ionizing radiation . In view of these findings we examined possible effects of PKCI on susceptibility to carcinogenicity . Both PKCI+/+ and PKCI-/- mice were treated with the chemical carcinogen N-nitrosomethylbenzylamine (NMBA) by intragastric administration and killed 12 weeks later . As expected with this protocol, NMBA induced squamous tumors (both papillomas and carcinomas) of the forestomach . The incidence, multiplicity per mouse, volume, and degree of malignancy of these tumors were significantly greater in the PKCI-/- than in the PKCI+/+ mice . Furthermore, four adenomas and one adenocarcinoma of the glandular stomach were found in the NMBA-treated PKCI-/- mice but no tumors of the glandular stomach were found in the NMBA-treated PKCI+/+ mice or in any of the untreated mice . Taken together, these findings suggest that, like FHIT, PKCI may normally play a tumor-suppressor role . The possible role of PKCI as a tumor suppressor in humans remains to be determined.

RNA, 2003 Jul, 9(7), 771 - 9
A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast; Badis G et al.; Ribosomal RNAs contain a number of modified nucleotides . The most abundant nucleotide modifications found within rRNAs fall into two types: 2'-O-ribose methylations and pseudouridylations . In eukaryotes, small nucleolar guide RNAs, the snoRNAs that are the RNA components of the snoRNPs, specify the position of these modifications . The 2'-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs, respectively . The role of these modifications in rRNA remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2'-O-ribose methylations or pseudouridylations . Only very recently, a slight translation defect and perturbation of polysome profiles was reported in yeast for the absence of the Psi at position 2919 within the LSU rRNA . Here we report the identification and characterization in yeast of a novel intronic H/ACA snoRNA that we called snR191 and that guides pseudouridylation at positions 2258 and 2260 in the LSU rRNA . Most interestingly, these two modified bases are the most conserved pseudouridines from bacteria to human in rRNA . The corresponding human snoRNA is hU19 . We show here that, in yeast, the presence of this snoRNA, and hence, most likely, of the conserved pseudouridines it specifies, is not essential for viability but provides a growth advantage to the cell.

Biochemistry, 2003 Jun 24, 42(24), 7487 - 96
Association of an aminoacyl-tRNA synthetase with a putative metabolic protein in archaea; Lipman RS et al.; Aminoacyl-tRNA synthetases are essential enzymes that catalyze attachment of amino acids to tRNAs for decoding of genetic information . In higher eukaryotes, several synthetases associate with non-synthetase proteins to form a high-molecular mass complex that may improve the efficiency of protein synthesis . This multi-synthetase complex is not found in bacteria . Here we describe the isolation of a non-synthetase protein from the archaeon Methanocaldococcus jannaschii that was copurified with prolyl-tRNA synthetase (ProRS) . This protein, Mj1338, also interacts with several other tRNA synthetases and has an affinity for general tRNA, suggesting the possibility of forming a multi-synthetase complex . However, unlike the non-synthetase proteins in the eukaryotic complex, the protein Mj1338 is predicted to be a metabolic protein, related to members of the family of H(2)-forming N(5),N(10)-methylene tetrahydromethanopterin (5,10-CH(2)-H(4)MP) dehydrogenases that are involved in the one-carbon metabolism of the archaeon . The association of Mj1338 with ProRS, and with other components of the protein synthesis machinery, thus suggests the possibility of a closer link between metabolism and decoding in archaea than in eukarya or bacteria.

J Biomed Mater Res B Appl Biomater, 2003 Jul 15, 66(1), 287 - 98
SEM and TEM analysis of water degradation of human dentinal collagen; Hashimoto M et al.; Recently several long-term studies have reported evidence of the hydrolytic degradation of collagen fibrils based on fractured surface observations after bond testing . Those studies suggested that one cause of the decline in the bond strength was the degradation of the collagen fibrils within the bonds . However, one concern has been raised that the dentinal collagen fibrils may be stable in water that does not contain oral bacteria or enzymes . Therefore, the present study aimed to clarify the micromorphological change in naked collagen fibrils after 500 days of water storage . To prepare exposed collagen fibrils, sectioned and polished human dentin surfaces were acid conditioned for 15 s with the use of two commercially available acid conditioners: All-Etch (10% phosphoric acid) and Uni-Etch (32% phosphoric acid) (Bisco, Inc.) . Those specimens were stored in distilled water at 37 degrees C for 1 day (control) for 500 days . After the storage periods, the samples were examined with the use of SEM and TEM . Under SEM and TEM examination, micromorphological alterations (disarrangement of collagen web, widening the interfibrillar space, and the thinning diameter of collagen fibrils) were found in the specimens after 500 days in water .

J Cell Sci, 2003 Jul 15, 116(Pt 14), 2839 - 44
Intramembrane proteolysis by presenilin and presenilin-like proteases; Xia W et al.; Regulated intramembrane proteolysis is a novel mechanism involving proteases that hydrolyze their substrates in a hydrophobic environment . Presenilin (PS) 1 and PS 2 are required for intramembrane cleavage of an increasing number of type I membrane proteins, including the amyloid precursor protein of Alzheimer's disease and the Notch receptor, which signals during differentiation and development . Mutagenesis, affinity labeling, biochemical isolation, and reconstitution in cells reveal that PS, in complex with co-factors nicastrin, APH-1 and PEN-2, apparently contains the active site of gamma-secretase, a novel membrane aspartyl protease . In addition, other related aspartyl proteases have been identified . These include members of the type-4 prepilin peptidase family in bacteria, which are known proteases and carry a GD motif conserved in PS . A group of multi-pass membrane proteins found in eukaryotes also contain YD and LGXGD motifs in two transmembrane domains that are conserved in PS and postulated to constitute an aspartyl protease active site . Among these is signal peptide peptidase (SPP), which cleaves remnant signal peptides derived from signal-peptidase-mediated ectodomain shedding . SPP cuts type II membrane proteins, illustrating that PS-like proteases play a key role in intramembrane proteolysis of single-pass membrane proteins oriented in either direction.

J Biol Chem, 2003 Aug 29, 278(35), 33450 - 5 Epub 2003 Jun 14.
Cell cycle-associated changes in Slingshot phosphatase activity and roles in cytokinesis in animal cells; Kaji N et al.; During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission . Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics . However, little is known about the mechanism controlling cofilin activity during cytokinesis . Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1) . Here we show that the phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation . SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody . Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells . Co-expression of cofilin rescued the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis . These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis.

J Biol Chem, 2003 Aug 22, 278(34), 32380 - 9 Epub 2003 Jun 13.
Characterization of mouse short-chain aldehyde reductase (SCALD), an enzyme regulated by sterol regulatory element-binding proteins; Kasus-Jacobi A et al.; Sterol regulatory element-binding proteins (SREBPs) enhance transcription of genes encoding all of the proteins required for the cellular synthesis and uptake of cholesterol and unsaturated fatty acids . Here, we use suppression subtractive hybridization to identify a previously unrecognized SREBP-enhanced gene in mice . The gene encodes a membrane-bound enzyme that we designate SCALD, for short-chain aldehyde reductase . We expressed SCALD in bacteria, purified it extensively, and studied its catalytic properties in detergent solution . The enzyme specifically uses NADPH to reduce a variety of short-chain aldehydes, including nonanal and 4-hydroxy-2-nonenal . The enzyme also reduces retinaldehydes, showing equal activity for all-trans-retinal and 9-cis-retinal . Northern blot analysis indicates that SCALD is expressed most abundantly in mouse liver and testis . In the liver of mice, SCALD is suppressed by fasting and induced by refeeding, consistent with regulation by SREBPs . In testis, SCALD expression is restricted to pachytene spermatocytes, as revealed by visualization of mRNA and protein . SCALD is also expressed in four layers of the retina, including the outer segment of rods and cones, as revealed by immunohistochemistry . SCALD appears to be the mouse ortholog of the human protein that has been designated variously as prostate short-chain dehydrogenase/reductase 1, retinal reductase 1, and retinol dehydrogenase 11 . In view of its ability to reduce short-chain aldehydes in addition to retinals, we propose that SCALD may be induced by SREBP in liver and other tissues to prevent toxicity from fatty aldehydes that are generated from oxidation of unsaturated fatty acids that are synthesized as a result of SREBP activity.

Immunology, 2003 Jul, 109(3), 384 - 91
Efficient antigen presentation of soluble, but not particulate, antigen in the absence of Wiskott-Aldrich syndrome protein; Westerberg L et al.; B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions . We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome . In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice . As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA . We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells . Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells . Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells . In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 795 - 800
Acidovorax valerianellae sp . nov., a novel pathogen of lamb's lettuce {Valerianella locusta (L.) Laterr.}; Gardan L et al.; Bacterial spot disease of lamb's lettuce {Valerianella locusta (L.) Laterr.} was first observed in fields in 1991 . This new bacterial disease is localized in western France in high-technology field production of lamb's lettuce for the preparation of ready-to-use salad . Nineteen strains isolated in 1992 and 1993 from typical black leaf spots of naturally infected lamb's lettuce were characterized and compared with reference strains of Acidovorax and Delftia . The pathogenicity of the 19 strains was confirmed by artificial inoculation . Biochemical and physiological tests, fatty acid profiles, DNA-DNA hybridization and other nucleic acid-based tests were performed . A numerical taxonomic analysis of the 19 lamb's lettuce strains showed a single homogeneous phenon closely related to previously described phytopathogenic taxa of the genus Acidovorax . DNA-DNA hybridization studies showed that the lamb's lettuce strains were 91-100% related to a representative strain, strain CFBP 4730(T), and constituted a discrete DNA hybridization group, indicating that they belong to the same novel species . Results from DNA-rRNA hybridization, 16S rRNA sequence analysis and fatty acid analysis studies confirmed that this novel species belongs to the beta-subclass of the Proteobacteria and, more specifically, to the family Comamonadaceae and the genus Acidovorax . The name Acidovorax valerianellae sp . nov . is proposed for this novel taxon of phytopathogenic bacteria . The type strain is strain CFBP 4730(T) (= NCPPB 4283(T)).

Kansenshogaku Zasshi, 2003 May, 77(5), 316 - 22
{Cytotoxic effect of antiseptics: comparison In vitro . In vivo examination of strong acidic electrolyzed water, povidone-iodine, chlorhexidine and benzalkonium chloride}; Iwasawa A et al.; Cytotoxic effect and guinea pig wound cure stage, pus fabrication presence in infected wound were compared with strong acidic electrolyzed water (AcEW) and povidone-iodine solution (PVP-I), chlorhexidine (CHG) and benzalkonium chloride (BAC) . It gave the following results: In a cytotoxic test, the toxicity was recognized in 0.1%-0.01% PVP-I, in 0.0002-0.0004% CHG, in 10-0.1 micrograms/ml BAC, but there was no toxicity in AcEW . By a guinea pig wound cure process, no significance was recognized between each pharmaceutical agent in epidermal cell migration, but by an inflammation locus area, the significance was considerable in comparison with no-treatment . The pyopoiesis of P . aeruginosa infected wound was recognized in a ratio of 38.2% physiological saline, 27.3% CHG, 20.6% PVP-I and 12.1% AcEW . When pollution locus includes an infection image of bacteria, while draining AcEW instead of physiological saline, disinfection, indication was expected, and, as for the disorder in cure stage . I do not agree with that mentioned above . As for AcEW, availability by organism use was recognized for the cytotoxic effect of antiseptic instead of action of acceleration for wound cure.

ScientificWorldJournal, 2003 May 28, 3, 432 - 42
A calorimetric characterization of Cr(VI)-reducing Arthrobacter oxydans at different phases of the cell growth cycle; Bakradze NG et al.; This is the first of a series of calorimetric studies designed to characterize and understand survival mechanisms of metal-reducing bacteria isolated from metal-polluted environments . In this paper we introduce a new concept of thermal spectrum of the endothermic melting of complex biological systems (e.g., proteins, nucleic acids, ribosomes, membrane structures) in intact cells . All thermal spectra measured are thermograms that describe the temperature dependence of heat capacity change of the complex systems of biologically active substances in bacterial cells . This new concept of thermal spectrum was applied to investigate spectral features from intact cells of Cr(VI)-reducer Arthrobacter oxydans at different points of their growth conditions and stages . Over the temperature range of 40-105 degrees C, we observed that spectral changes are particularly significant in the 40-90 degrees C interval . This may correspond to the orderly changes in subcellular structural elements: proteins, ribosomes and RNA, membranes, and various structural elements of the cell wall during different points of the growth cycle and growth conditions . Spectral changes in the 90-105 degrees C region are less pronounced, implicating that the structural composition of DNA-Protein (DNP) complexes may change little.

ScientificWorldJournal, 2002 Feb 05, 2, 308 - 19
Mobilisation and bioavailability of arsenic around mesothermal gold deposits in a semiarid environment, Otago, New Zealand; Craw D et al.; Arsenopyrite (FeAsS) is the principal arsenic (As) mineral in mineralised mesothermal veins (typically 5,000 mg/kg As) in southeastern New Zealand . Groundwater in contact with arsenopyrite-bearing rocks has elevated As concentrations (up to 0.1 mg/l) . The arsenopyrite decomposes slowly on oxidation in soils and historic mine workings in a cool semiarid climate . Dissolved As is predominantly As(III) in association with arsenopyrite, but this is rapidly oxidised over days to weeks to As(V) in the vadose zone . Oxidation is facilitated by particulate Fe and/or Mn oxyhydroxides, and by bacteria in surface waters . Evaporative concentration of dissolved As(V) in the vadose zone causes precipitation of scorodite (Fe(III)As(V)O4*2H2O) . Adsorption of As(V) to Fe oxyhydroxides in soils and groundwater pathways lowers dissolved As concentrations . Soils over mineralised veins typically have <200 mg/kg As, as most As is removed in solution on geological time scales . Most plants on the mineralised rocks and soils do not take up As, although some inedible species can fix up to 18 mg/kg As . Hence, bioavailability of As(V) is low in this environment, despite the substantial As flux . Similar As mobility is seen in an active gold mine processing plant and tailings . Arsenopyrite dissolves more rapidly on agitation, and mine waters can have dissolved As >200 mg/l, predominantly as As(V) . This dissolved As decreases in tailings waters to near 2 mg/l, mainly as As(III) when in contact with arsenopyrite . Weak oxidation of evaporatively dried tailings causes cementation with scorodite and iron oxyhydroxides, and scorodite precipitation exerts some control on dissolved As(V) concentrations . High dissolved As in mine waters is lowered by adsorption to iron oxyhydroxides, and waters discharged from the mine site have negligible dissolved As.

ScientificWorldJournal, 2002 Apr 08, 2, 926 - 42
Zooplankton structure and potential food web interactions in the plankton of a subtropical chain-of-lakes; Havens KE; This study evaluates the taxonomic and size structure of macro-zooplankton and its potential role in controlling phytoplankton in the Kissimmee Chain-of-Lakes, six shallow interconnected lakes in Florida, U.S . Macro-zooplankton species biomass and standard limnological attributes (temperature, pH, total phosphorus {TP}, chlorophyll a {Chl a}, and Secchi transparency) were quantified on a bimonthly basis from April 1997 to February 1999 . Concentrations of TP ranged from below 50 to over 150 microg l(-1) . Peak concentrations of particulate P coincided with maximal Chl a, and in one instance a high concentration of soluble reactive P followed . The cladoceran zooplankton was dominated by small species, including Eubosmina tubicen, Ceriodaphnia rigaudi, and Daphnia ambigua . The exotic daphnid, D . lumholtzii, periodically was abundant . The copepods were strongly dominated by Diaptomus dorsalis, a species previously shown to be highly resistant to fish predation . These results, and findings of controlled experiments on a nearby lake with a nearly identical zooplankton species complement, suggest that fish predation may be a major factor structuring the macro-zooplankton assemblage . Zooplankton biomass, on the other hand, may be affected by resource availability . There was a significant positive relationship between average biomass of macro-zooplankton and the average concentration of TP among the six lakes . No such relationship existed between zooplankton biomass and Chl a, suggesting that the predominant food web in these systems may be based on bacteria-plankton, as has been documented in nearby Lake Okeechobee . All of the zooplankton taxa encountered in the Kissimmee Chain-of-Lakes (except Mesocyclops edax) are known bacteria grazers in Florida lakes . Phytoplankton biomass, measured as Chl a, was strongly associated with TP, both within and across lakes . Phytoplankton biomass was not associated with the biomass of zooplankton . These results, when considered in the context of nutrient-addition, zooplankton-exclosure studies on Lake Okeechobee, support the hypothesis that phytoplankton biomass in subtropical lakes is regulated by "bottom-up," rather than "top-down" forces.

Science, 2003 Jun 13, 300(5626), 1706 - 7
Phylogenomics: intersection of evolution and genomics; Eisen JA et al.; Much has been gained from genomic and evolutionary studies of species . Combining the perspectives of these different approaches suggests that an integrated phylogenomic approach will be beneficial.

Mol Cancer Res, 2003 Jun, 1(8), 598 - 609
Regulation of the Chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation; Schwarz JK et al.; Chk2 is a serine/threonine protein kinase found mutated in certain hereditary and sporadic cancers . Ionizing radiation (IR) activates the kinase activity of Chk2 in a phosphorylation-dependent manner . ATM phosphorylates Chk2 on threonine 68, which promotes oligomerization and phosphorylation on threonines 383 and 387 within the activation loop of the catalytic domain . In this study, threonines 68, 383, and 387 were confirmed as sites of Chk2 phosphorylation both in vitro and in vivo . In addition, serine 516 was identified as a novel IR-inducible phosphorylation site in vivo and as a site of autophosphorylation in vitro . Interestingly, Chk2 was capable of autoactivation in the absence of IR when overproduced in bacteria, in 293 cells, and in murine embryonic fibroblasts lacking Chk2 . A kinase-inactive mutant of Chk2 was phosphorylated on T68 and T383/T387 but not on S516 in cells containing Chk2 and on T68 but not T383/T387 or S516 in cells lacking Chk2 . This establishes a dependency on Chk2 kinase activity for phosphorylation of T383/T387 and S516 but not for T68 in vivo . We demonstrate that T68 phosphorylation is regulated by kinases in addition to ATM and Chk2 . Taken together, our data indicate that autophosphorylation of Chk2 can occur both in cis and in trans and suggest that oligomerization may regulate Chk2 activation by promoting these cis- and trans-phosphorylation events . The importance of oligomerization is underscored by the observation that substitution of isoleucine for threonine at position 157, a mutation found in a subset of patients with Li-Fraumeni syndrome, impairs both Chk2 oligomerization and autophosphorylation.

FEBS Lett, 2003 Jun 19, 545(2-3), 97 - 102
Are syn-ligated (bacterio)chlorophyll dimers energetic traps in light-harvesting systems?
Balaban TS.
A recent study of the stereochemical details of chlorophyll ligation in photosystem I {Balaban et al., Biochim . Biophys . Acta 1556 (2002) 197-207} has revealed that only 14 chlorophylls out of the total 96 are ligated from the same side (syn) as the 17-propionic acid residue which is esterified with phytol . The syn chlorophylls are carefully surrounding the reaction center forming the inner core antenna system and their ligands have been strongly conserved in several species during evolution . We hypothesize here that the two dimers of closely spaced syn chlorophylls which are encountered within roughly 2 nm of P700 are the ultimate energetic traps of this light-harvesting system . Structurally very similar bacteriochlorophyll a dimers are encountered within the Fenna-Matthews-Olson protein complex and within the B850 ring of the LH2 complex of purple bacteria . The non-random disposal of these dimers lends support to our hypothesis that the syn ligation coupled with a strong excitonic interaction leads to the most red-shifted pigments in light-harvesting systems . We would like to encourage both theoretical and experimental studies to either prove or disprove this intriguing structure-function conjecture in view of designing efficient artificial light-harvesting systems.

Cochrane Database Syst Rev . 2003;(2):CD003875.
Enamel matrix derivative (Emdogain) for periodontal tissue regeneration in intrabony defects; Esposito M et al.; BACKGROUND: Periodontitis is a chronic infective disease of the gums caused by bacteria present in dental plaque . This condition induces the breakdown of the tooth supporting apparatus until teeth are lost . Surgery may be indicated to arrest disease progression and regenerate lost tissues . Several surgical techniques have been developed to regenerate periodontal tissues including guided tissue regeneration (GTR), bone grafting (BG) and the use of enamel matrix derivative (EMD) . EMD is an extract of enamel matrix and contains amelogenins of various molecular weights . There is evidence to show that amelogenins are involved not only in enamel formation, but also in the formation of the periodontal attachment during tooth formation . OBJECTIVES: To test the efficacy of EMD in comparison with open flap debridement, GTR and various BG procedures for the treatment of intrabony defects . SEARCH STRATEGY: We searched the Cochrane Oral Health Group's Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE . Several journals were handsearched . No language restrictions were applied . Authors of randomised controlled trials (RCTs) identified, personal contacts and the manufacturer were contacted to identify unpublished trials . Most recent search: January 2003 . SELECTION CRITERIA: RCTs on patients affected by periodontitis having intrabony defects treated with EMD compared with open flap debridement, GTR and various BG procedures with at least one year follow up . The outcome measures considered were: tooth loss, changes in probing attachment levels (PAL), pocket depths (PPD), gingival recessions (REC), marginal bone levels on intraoral radiographs and postoperative infections . DATA COLLECTION AND ANALYSIS: Screening of eligible studies, assessment of the methodological quality of the trials and data extraction were conducted in duplicate and independently by two reviewers . Results were expressed as random effect models using weighted mean differences for continuous outcomes and relative risk for dichotomous outcomes with 95% confidence interval (CI) . Heterogeneity was investigated including both clinical and methodological factors . MAIN RESULTS: No difference in tooth loss was observed . A meta-analysis including eight trials showed that Emdogain treated sites displayed statistically significant PAL improvements (mean difference 1.3 mm, 95%CI: 0.8 to 1.8) and PPD reduction (1 mm, 95%CI: 0.5 to 1.4) when compared to flap surgery . Comparing Emdogain with GTR (six trials), GTR showed a statistically significant reduction of PPD (0.6 mm) and increase of REC (0.5 mm) . No difference in postoperative infections was observed . REVIEWER'S CONCLUSIONS: Emdogain is able to significantly improve PAL levels (1.3mm) and PPD reduction (1mm) when compared to flap surgery, however these results may not have a great clinical impact, since it has not been shown that more periodontally compromised teeth could be saved . There was no evidence of clinically important differences between GTR and Emdogain.

Mol Ecol, 2003 Jul, 12(7), 1843 - 54
Wolbachia and genetic variability in the birdnest blowfly Protocalliphora sialia; Baudry E et al.; Wolbachia are widespread cytoplasmically inherited bacteria that induce various reproductive alterations in host arthropods, including cytoplasmic incompatibility (CI), an incompatibility between sperm and egg that typically results in embryonic death . CI has been invoked as a possible mechanism for reproductive isolation and speciation in arthropods, by restricting gene flow and promoting maintenance (and evolution) of genetic divergence between populations . Here we investigate patterns of Wolbachia infection and nuclear and mitochondrial differentiation in geographical populations of the birdnest blowfly Protocalliphora sialia . Blowflies in western North America are infected with two A-group Wolbachia, with some individuals singly and others doubly infected . Individuals in eastern North America mostly show single infections with a B-group Wolbachia . Populations in the Midwest are polymorphic for infections and show A- or B-group infection . There is a low level of mitochondrial divergence and perfect concordance of mitochondrial haplotype with infection type, suggesting that two Wolbachia-associated selective sweeps of the mitochondrion have occurred in this species . Amplified fragment length polymorphism analysis of nuclear genetic variation shows genetic differentiation between the eastern-Midwestern and western populations . Both Midwestern and eastern flies infected with A-Wolbachia show eastern nuclear genetic profiles . Current results therefore suggest that Wolbachia has not acted as a major barrier to gene flow between western and eastern-Midwestern populations, although some genetic differentiation between A-Wolbachia infected and B-Wolbachia infected individuals in eastern-Midwestern populations cannot be ruled out.

Mol Ecol, 2003 Jul, 12(7), 1757 - 71
Effects of Wolbachia on mtDNA variation in two fire ant species; Dewayne Shoemaker D et al.; Wolbachia are endosymbiotic bacteria that infect arthropods . As they are maternally transmitted, the spread of Wolbachia variants within host populations may affect host mtDNA evolution . We sequenced a portion of the mitochondrial cytochrome oxidase I gene from numerous individuals of two Wolbachia-infected fire ant species, Solenopsis invicta and S . richteri, to determine how these bacteria influence patterns of mtDNA variation . As predicted, there was a strong association between Wolbachia strain and host mtDNA lineage within and between these fire ant species . However, there was no consistent association between the presence of Wolbachia and a reduction in mtDNA diversity . Moreover, patterns of mtDNA variation within Wolbachia-infected populations did not differ consistently from neutral expectations, despite our prediction that strong positive selection acting on Wolbachia influences the evolutionary dynamics of other cytoplasmic genomes . Specifically, while values of Tajima's D consistently were less than zero for all six samples of fire ants harbouring Wolbachia, MacDonald-Kreitman tests suggested that the patterns of variation were different from those expected under neutrality in only two of the samples . We conclude that these neutrality tests do not unambiguously reveal a clear effect of Wolbachia infection on patterns of mtDNA variation and substitution in fire ants . Finally, consistent with an earlier study, our data revealed the presence of two divergent mtDNA haplotype lineages and Wolbachia strains within S . invicta . Recognition of these two lineages has important consequences for interpreting patterns of mtDNA evolution and genetic differentiation between conspecific social forms of this species.

Fundam Clin Pharmacol, 2003 Jun, 17(3), 301 - 13
Gastrointestinal effects of nonsteroidal anti-inflammatory drugs; Whittle BJ; Non-steroidal anti-inflammatory drugs (NSAIDs) causes extensive damage to the gastrointestinal (GI) tract . The underlying mechanisms of gastric injury include topical irritant actions that disrupt the epithelial barrier, as well as the inhibition of cyclo-oxygenase (COX), which is predominantly the COX-1 isoform in the mucosa . This damage can be attenuated by antisecretory agents or by mucosal protective agents such as the synthetic prostanoids or nitric oxide (NO) donors . Compounds designed to attenuate topical irritancy, or have protective agents incorporated, such as NO-containing NSAIDs, the CINODs (cyclo-oxygenase-inhibiting NO-donating drugs) show reduced mucosal injury . NSAIDs also cause injury in the small intestine, which appears to result from initial COX inhibition, with subsequent translocation of indigenous bacteria, induction of NO synthase and production of the cytotoxic moiety, peroxynitrite . The COX-2 selective agents, the coxibs, which inhibit prostanoid biosynthesis at inflammatory sites, but not the endogenous protective prostanoids in the gut formed by COX-1, have proved so far to be a successful therapeutic approach to reducing NSAIDs GI damage . The clinical outcome of the use of the second generation of coxibs, and the newer NO NSAIDs is now awaited.

Biochem J, 2003 Sep 15, 374(Pt 3), 577 - 606
RecQ helicases: suppressors of tumorigenesis and premature aging; Bachrati CZ et al.; The RecQ helicases represent a subfamily of DNA helicases that are highly conserved in evolution . Loss of RecQ helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination . Germ-line defects in three of the five known human RecQ helicases give rise to defined genetic disorders associated with cancer predisposition and/or premature aging . These are Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome, which are caused by defects in the genes BLM, WRN and RECQ4 respectively . Here we review the properties of RecQ helicases in organisms from bacteria to humans, with an emphasis on the biochemical functions of these enzymes and the range of protein partners that they operate with . We will discuss models in which RecQ helicases are required to protect against replication fork demise, either through prevention of fork breakdown or restoration of productive DNA synthesis.

Environ Technol, 2003 May, 24(5), 627 - 40
Factors affecting washout in intermittently feed upflow anaerobic reactors: experimental tests and modelling; Garuti G et al.; This paper reports the results of experimental investigations carried out on a full-scale ABR and on a pilot UASB to determine biomass washout for various intermittent feed conditions . Particularly, the tests aimed to determine the extent to which sludge washout is affected by such factors as: daily flow distribution; upflow velocity; concentration and sedimentation properties of the biomass . Moreover, in order to obtain a tool to estimate sludge washout in different influent flow conditions, a simulation model of the sludge transport phenomena in the uplow anaerobic reactors is proposed . The results showed that when the influent flow is interrupted for long periods (above 1 hour), once it is resumed considerable losses of biomass into the effluent were found, independent of the upflow velocity . In contrast with shorter periods, regular sludge expansion took place even with very high upflow velocities (up to 4 m h(-1)), giving rise to limited sludge washout and high removal efficiencies.

Environ Technol, 2003 May, 24(5), 573 - 87
Effects of trace element addition on volatile fatty acid conversions in anaerobic granular sludge reactors; Osuna MB et al.; The effect of the addition of trace elements on the conversion of a mixture of volatile fatty adds (Acetate, Propionate, Butyrate, in a ratio 3:1:1) by anaerobic granular sludge was investiated . Two Upflow Anaerobic Sludge Bed reactors (pH 7, ranging 30 degrees C) were operated for 140 days at an organic loading rate from 2 g COD l(-1) d(-1) up to 10 g COD l(-1) d(-1) and a hydraulic retention time of 12 hours . One reactor (R1) was supplied with a trace metal cocktail in the basal medium, whereas trace metals were omitted from the influent of the second reactor (R2) . As a result, the trace metal concentration in the granules from R2 steadily decreased at a rate of 48 microg metal g(-1) TS d(-1) down to 35% of their initial value . In contrast, trace metals accumulated in granules present in R1 . At the end of the experiment, the COD removal efficiencies were 99 and 77% for, respectively, the control (R1) and deprived (R2) reactors . This difference was due to lack of propionate conversion by sludge from R2 . No difference in the acetate and butyrate conversion capacity of both reactors was observed . The conversion of acetate, propionate and methanol were stimulated by the continuous addition of metals to the influent, a sludge of R1 had higher maximum specific activity values compared to sludge of R2 . However, both sludges had a similar maximum specific activity with butyrate . Surprisingly, maximum specific activity tests using individual trace metals showed that the addition of a particular trace element in the activity test medium did not affect the degradation rates of aspecific substrate, i.e . acetate, propionate, butyrate and methanol.

Environ Technol, 2003 May, 24(5), 553 - 60
Impact of landfill leachate on anaerobic digestion of sewage sludge; Hombach ST et al.; The feasibility of mesophilic anaerobic co-digestion of landfill leachate and sewage sludge was examined in a bench-scale experiment . Three complete-mix, flow-through digesters were operated in a semi-continuous mode . During both phases of research all digesters received 500 ml d(-1) of raw sludge and Reactor 1 was always the control reactor--fed sludge only . During Phase 1, leachate volumes less than 12% of the sludge volume were fed to Reactors 2 and 3 . During Phase 2 larger amounts of leachate were added, exceeding 20% of sludge volume which led to an overall decrease in the hydraulic residence time of the digesters . All reactors achieved stable operation, which indicated that the co-digestion of sewage sludge and landfill leachate is feasible During Phase 1, an increase in the average daily methane production from 2.5 l d(-1) to 3.1 l d(-1) and 3.2 l d(-1) was observed; the biomethanation production (BMP) increased from 0.46 to 0.6 m3 - 0.7 m3 CH4 (kg VS rem.)(-1) . The average volatile solids reduction (VSR) increased from 46.1% to 48.6% and 49.0% . In Phase 2, the total methane production in the control reactor was significantly higher, at 4.6 l d(-1), while the addition of larger, by volume, amounts of leachate, decreased the methane production to 4.3 l d(-1) and 4.2 l d(-1), respectively . The average BMP values were 0.8, 0.87, and 0.81 m3 CH4 (kg VS rem.)(-1), respectively . In Phase 2, leachate addition decreased the average VSR from 51% to 49% and 45.6% . After calculating that leachate addition to digesters would not increase heavy metal concentrations in the produced biosolids it was concluded that mesophilic anaerobic co-digestion of sewage sludge and landfill leachate is feasible, and provides a promising alternative to aerobic co-treatment.

Eye, 2003 May, 17(4), 506 - 12
Residual debris as a potential cause of postphacoemulsification endophthalmitis; Leslie T et al.; AIM: To examine residual debris within sterilised instruments prior to cataract surgery . METHODS: (i) Flushings from 32 sets of phacoemulsification instruments, sterilised according to hospital routine protocols, were taken preoperatively and analysed by scanning electron microscopy (SEM) . (ii) A total of 16 sets of flushings from a different institute were collected-with separation of samples collected from phacoemulsification and those from irrigation-aspiration (IA) instruments-and analysed in the same way . (iii) A total of 15 sets of flushings were collected from instruments where an automated flushing system was used prior to sterilisation . RESULTS: (i)In the first study, 62% were clean, 16% were moderately contaminated and 22% were severely contaminated . Various contaminants were identified including lens capsule and cells, man-made fibres, squamous cells, bacteria, fungal elements, diatoms, red blood cells and proteinaceous material . (ii) In the second study, the results were similar and contamination of both phacoemulsification and IA instruments was shown . (iii) The third study showed that although a decrease in contamination followed automated flushing, contamination was not completely eliminated . CONCLUSIONS: Although all equipment had been sterilised, pyrogenic material was still present . These findings emphasise the importance of meticulous cleaning of all surgical equipment in which biological debris can remain.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7720 - 5 Epub 2003 Jun 11.
piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila; Hacker U et al.; P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability . Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration . In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system . These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents . To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons . An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein . In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit . The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes . PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported . Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome . The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms.

Gut, 2003 Jul, 52(7), 1054 - 9
Increased tumour necrosis factor alpha production in mesenteric lymph nodes of cirrhotic patients with ascites; Genesca J et al.; BACKGROUND: Cytokines produced in mesenteric lymph nodes of cirrhotic rats with bacterial translocation may participate in circulatory alterations of cirrhosis . AIM: To investigate whether cirrhotic patients present an increased local generation of cytokines in mesenteric lymph nodes . METHODS: Mesenteric lymph nodes from 26 cirrhotic and 10 control patients were assessed for tumour necrosis factor alpha (TNF) and interleukin 6 mRNA and protein expression by competitive reverse transcription-polymerase chain reaction, and by enzyme immunoassay and immunohistochemistry, respectively . RESULTS: Interleukin 6 levels were not different between cirrhotics and controls . Protein and mRNA TNF levels in mesenteric lymph nodes from cirrhotics were higher than in controls (p<0.05) . Tissue expression of TNF by immunohistochemistry was more abundant in cirrhotics . Ascitic patients showed higher TNF levels (47 (34-54) pg/mg protein) than patients without ascites (18 (17-25) pg/mg protein) (p<0.001) . Elevated TNF levels (>28 pg/mg protein) in cirrhotics were associated with a higher Child-Pugh score, the antecedent of ascites, a lower prothrombin rate, and higher bilirubin and blood TNF levels . The strongest association, confirmed by multivariate analysis, was with the presence of ascites (p<0.001) . Bacterial infections after transplantation, mainly by enteric bacteria, were only detected in patients with high TNF levels in mesenteric lymph nodes (33% of patients; p=0.05) . CONCLUSION: Patients with advanced liver cirrhosis, and especially with ascites, have increased local production of TNF in mesenteric lymph nodes that, in common with experimental cirrhosis, may also be induced by bacterial translocation.

Gut, 2003 Jul, 52(7), 933 - 7
13C urea breath test (UBT) in the diagnosis of Helicobacter pylori: why does it work better with acid test meals?
Pantoflickova D, Scott DR, Sachs G, Dorta G, Blum AL.
BACKGROUND: Acid test meals may improve the accuracy of the (13)C urea breath test (UBT) . This has been attributed to changes in gastric emptying rather than to the effects of gastric pH on Helicobacter pylori urease . AIMS: To determine whether enhancement of (13)CO(2) excretion in the UBT in H pylori infected volunteers by acidification of a test meal is due to a delay in gastric emptying . METHODS: Urease activity in vitro was measured in intact bacteria and in bacterial homogenates . Urease activity in vivo was assessed by means of the UBT . Eleven H pylori infected subjects underwent UBTs with neutral Ensure (pH 7.0), acidified Ensure (pH 3.0), and apple juice (pH 3.0) . Gastric emptying was assessed by (13)C sodium acetate breath test . RESULTS: From pH 7 to pH 3, the in vitro urease activity of intact bacteria increased sixfold . In contrast, urease activity of bacterial homogenates was inactivated by low pH . In vivo, urease activity, as measured by the UBT 20 minutes after meal ingestion, was higher with apple juice (delta (13)CO(2)=21.1; p=0.03) and acidified Ensure (delta (13)CO(2)=25.5; p=0.01) than with neutral Ensure (delta (13)CO(2)=12.5) . Gastric emptying was faster with apple juice (T(max)=36.7 (8) minutes) but not with acidified Ensure (T(max)=63.3 (5) minutes; p=0.06) than with neutral Ensure (T(max)=65.0 (3) minutes; p=0.04) . CONCLUSIONS: The higher UBT found with acidified compared with neutral test meals was independent of the emptying rates of the test meals but may have been due to medium acidity dependent activation of intra-bacterial urease in intact H pylori.

Lancet, 2003 Jun 7, 361(9373), 1939 - 44
Personal exposure to nitrogen dioxide (NO2) and the severity of virus-induced asthma in children; Chauhan AJ et al.; BACKGROUND: A link between exposure to the air pollutant nitrogen dioxide (NO2) and respiratory disease has been suggested . Viral infections are the major cause of asthma exacerbations . We aimed to assess whether there is a relation between NO2 exposure and the severity of asthma exacerbations caused by proven respiratory viral infections in children . METHODS: A cohort of 114 asthmatic children aged between 8 and 11 years recorded daily upper and lower respiratory-tract symptoms, peak expiratory flow (PEF), and measured personal NO2 exposures every week for up to 13 months . We took nasal aspirates during reported episodes of upper respiratory-tract illness and tested for infection by common respiratory viruses and atypical bacteria with RT-PCR assays . We used generalised estimating equations to assess the relation between low (<7.5 microg/m3), medium (7.5-14 microg/m3 ), and high (>14 microg/m3) tertiles of NO2 exposure in the week before or after upper respiratory-tract infection and the severity of asthma exacerbation in the week after the start of an infection . FINDINGS: One or more viruses were detected in 78% of reported infection episodes, and the medians of NO2 exposure were 5 (IQR 3.6-6.3), 10 (8.7-12.0), and 21 microg/m3 (16.8-42.9) for low, medium, and high tertiles, respectively . There were significant increases in the severity of lower respiratory-tract symptom scores across the three tertiles (0.6 for all viruses {p=0.05} and >2 for respiratory syncytial virus {p=0.01}) and a reduction in PEF of more than 12 L/min for picornavirus (p=0.04) for high compared with low NO2 exposure before the start of the virus-induced exacerbation . INTERPRETATION: High exposure to NO2 in the week before the start of a respiratory viral infection, and at levels within current air quality standards, is associated with an increase in the severity of a resulting asthma exacerbation.

Gene, 2003 May 22, 310, 59 - 66
Transfer RNA paralogs: evidence for genetic code-amino acid biosynthesis coevolution and an archaeal root of life; Xue H et al.; A search has been performed on 2878 tRNA sequences from 60 different genomes in order to detect the existence of closely related 'alloacceptor' tRNAs accepting dissimilar amino acids that could be paralogs generated by gene duplications . This has led to the identification of extremely conserved tRNA(Phe)-tRNA(Tyr) pairs displaying as high as 94% identity between them, and also other potentially paralogous tRNA pairs in archaeal species . These paralogous pairs are enriched for amino acid pairs belonging to the same amino acid biosynthetic family, thus providing evidence for the coevolution of genetic code and amino acid biosynthesis . Overall, the genetic distances between alloacceptor tRNAs yield estimates of how closely clustered in sequence space are the tRNAs in a genome . Among 34 Bacteria, 18 Archaea and 8 Eukarya, Methanopyrus kandleri and Aeropyrum pernix have yielded the lowest alloacceptor distances and largest number of paralogous pairs . Based on a cluster-dispersion model of tRNA evolution, such tight alloacceptor clustering is a measure of primitiveness of tRNA genotypes, and places last universal common ancestor (LUCA) between the branches leading to these two archaea in the tRNA phylogenetic tree.

Filaria J . 2003 May 9;2(1):10.
Obligatory symbiotic Wolbachia endobacteria are absent from Loa loa; Buttner DW et al.; BACKGROUND: Many filarial nematodes harbour Wolbachia endobacteria . These endobacteria are transmitted vertically from one generation to the next . In several filarial species that have been studied to date they are obligatory symbionts of their hosts . Elimination of the endobacteria by antibiotics interrupts the embryogenesis and hence the production of microfilariae . The medical implication of this being that the use of doxycycline for the treatment of human onchocerciasis and bancroftian filariasis leads to elimination of the Wolbachia and hence sterilisation of the female worms . Wolbachia play a role in the immunopathology of patients and may contribute to side effects seen after antifilarial chemotherapy . In several studies Wolbachia were not observed in Loa loa . Since these results have been doubted, and because of the medical significance, several independent methods were applied to search for Wolbachia in L . loa . METHODS: Loa loa and Onchocerca volvulus were studied by electron microscopy, histology with silver staining, and immunohistology using antibodies against WSP, Wolbachia aspartate aminotransferase, and heat shock protein 60 . The results achieved with L . loa and O . volvulus were compared . Searching for Wolbachia, genes were amplified by PCR coding for the bacterial 16S rDNA, the FTSZ cell division protein, and WSP . RESULTS: No Wolbachia endobacteria were discovered by immunohistology in 13 male and 14 female L . loa worms and in numerous L . loa microfilariae . In contrast, endobacteria were found in large numbers in O . volvulus and 14 other filaria species . No intracellular bacteria were seen in electron micrographs of oocytes and young morulae of L . loa in contrast to O . volvulus . In agreement with these results, Wolbachia DNA was not detected by PCR in three male and six female L . loa worms and in two microfilariae samples of L . loa . CONCLUSIONS: Loa loa do not harbour obligatory symbiotic Wolbachia endobacteria in essential numbers to enable their efficient vertical transmission or to play a role in production of microfilariae . Exclusively, the filariae cause the immunopathology of loiasis is patients and the adverse side effects after antifilarial chemotherapy . Doxycycline cannot be used to cure loiais but it probably does not represent a risk for L . loa patients when administered to patients with co-infections of onchocerciasis.

ANZ J Surg, 2003 Jun, 73(6), 453 - 9
Foundation lecture . Fracture care of friend and foe during World War I; Kirkup J; BACKGROUND: In the early years of trench warfare, compound lower limb fractures caused by gunshot missiles prompted the -questioning of traditional splintage techniques and established evacuation methods . These prejudiced recovery and delayed surgery, often by many days, causing a high mortality rate especially for open femoral fractures . Importantly, battle weaponry was modified by differences in climate and terrain in Mesopotamia, Gallipoli, the Alps and the Northern European plain, to manifest differences in wound pathology . In Flanders, the static situation limited simple bullet wounds and launched a high percentage of jagged shell -fragment injuries complicated by tissue destruction, in-driven clothing and metal contaminated with mud, lethal bacteria and spores . From no-mans-land, soldiers with arm fractures scrambled back unaided, with tibial fractures they might hobble between two comrades, but with femoral fractures they were helpless unless stretcher bearers arrived . Often they did not, or only after a lull in fighting, by which time death from blood loss or exposure supervened . Even on a stretcher, poor fracture immobilization and long arduous carries added to shock and mortality . Remedies to these deficiencies and observations by Australasian, Austrian, British, Canadian, French, German, South African and American surgeons are noted . CONCLUSIONS: Trained to treat bullet wounds in open terrain, many military medical organizations were slow to adjust to the novel challenges associated with trench warfare . However, from 1917 well-trained stretcher bearers, efficient application of the Thomas splint, better control of haemorrhage and more rapid evacuation with motorized ambulances reduced deaths, amputation rates and long-term disability significantly.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7913 - 8 Epub 2003 Jun 10.
A newly discovered cholesteryl galactoside from Borrelia burgdorferi; Ben-Menachem G et al.; Two major glycolipids, which comprise approximately 36% of the total lipid mass from Borrelia burgdorferi, the etiological agent of Lyme disease, were investigated . We determined the fatty acid type, sugar identity, anomeric configuration, and substituent type and position . The structures were identified as cholesteryl 6-O-acyl-beta-d-galactopyranoside (B . burgdorferi glycolipid 1, BbGL-I), and 1,2-di-O-acyl-3-O-alpha-d-galactopyranosyl-sn-glycerol (BbGL-II) . The major fatty acids were palmitate and oleate . The structures were corroborated by gas-liquid chromatography MS, matrix-assisted laser desorption/ionization time-of-flight spectroscopy, fast atom bombardment MS, detailed NMR spectrometry, and metabolic labeling . This is a previously undescribed demonstration of a cholesteryl galactoside in bacteria . Lipopolysaccharide was not detected in B . burgdorferi . The two glycolipids have several properties suggesting they may function as lipopolysaccharide: both are main components of the bacterial membrane, surface exposed, and have a three-domain structure . BbGL-I elicited specific antibodies in mice and rabbits, and BbGL-II elicited antibodies that reacted with both glycolipids.

Genome Res, 2003 Jun, 13(6A), 1244 - 9
Internet Contig Explorer (iCE)--a tool for visualizing clone fingerprint maps; Fjell CD et al.; Fingerprinted clone physical maps have proven useful in various applications, supporting both whole-genome and region-specific DNA sequencing as well as gene cloning studies . Fingerprint maps have been generated for several genomes, including those of human, mouse, rat, the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, Arabidopsis thaliana and rice . Fingerprint maps of other genomes, including those of fungi, bacteria, poplar, and the cow, are being generated . The increasing use of fingerprint maps in genomic research has spawned a need in the research community for intuitive computer tools that facilitate viewing of the maps and the underlying fingerprint data . In this report we describe a new Java-based application called iCE (Internet Contig Explorer) that has been designed to provide views of fingerprint maps and associated data . Users can search for and display individual clones, contigs, clone fingerprints, clone insert sizes and markers . Users can also load into the software lists of particular clones of interest and view their fingerprints . iCE is being used at our Genome Centre to offer up to the research community views of the mouse, rat, bovine, C . briggsae, and several fungal genome bacterial artificial chromosome (BAC) fingerprint maps we have either completed or are currently constructing . We are also using iCE as part of the Rat Genome Sequencing Project to manage our provision of rat BAC clones for sequencing at the Human Genome Sequencing Center at the Baylor College of Medicine.

Eur J Pharmacol, 2003 Jun 6, 470(3), 193 - 9
Therapeutic treatment with heat-killed Mycobacterium vaccae (SRL172) in a mild and severe mouse model for allergic asthma; Smit JJ et al.; The hypothesis that a lack of early childhood bacterial infections would favor the development of allergic disease suggests that bacteria can be used as a potential treatment for allergic asthma . Therefore, in this study, we investigated the therapeutic potential of heat-killed Mycobacterium vaccae in two mouse models of allergic asthma . For this purpose, mice were sensitized i.p . with ovalbumin/alum (severe model) or ovalbumin alone (mild model) and challenged on days 77, 80 and 83 by inhalation of either ovalbumin or saline aerosols . Treatment of mice with M . vaccae (s.c . 10(7) or 10(8) colony-forming units) on days 56 and 63, however, did not reduce airway hyperresponsiveness and eosinophilia, IgE and interleukin-5 production 24 h after ovalbumin challenge in either mouse model . We therefore conclude that treatment of sensitized mice with M . vaccae before allergen exposure is not able to reduce the allergic and asthma-like response in a mild and a severe model of allergic asthma.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Jun, 135(2), 297 - 308
Phospholipid fatty acids and sterols of two Cinachyrella sponges from the Saudi Arabian Red Sea: comparison with Cinachyrella species from other origins; Barnathan G et al.; Phospholipid class compositions, fatty acids and sterols of the sponges Cinachyrella alloclada and C . kukenthali from the Saudi Arabian Red Sea were studied and compared with previous results for other Cinachyrella spp . collected in Senegal (East Atlantic) and New Caledonia (West Pacific) . More than 50 fatty acids were identified as methyl esters and N-acyl pyrrolidides in each phospholipid mixture by GC/MS . Six fatty acids not hitherto found in nature were identified, namely 17-methyltetracosanoic in C . kukenthali and 18-methyltetracosanoic, 18-methylpentacosanoic, 18-methylhexacosanoic, 18,24-dimethyl-hexacosanoic and 6-bromo-5,9-nonacosadienoic acids in C . alloclada . Approximately 20 Delta 5,9 unsaturated fatty acids were found, including three 6-brominated acids . The presence of bacteria was evidenced by the relatively high proportions of phosphatidylglycerol and high levels of branched short-chain fatty acids . A total of 20 free 3beta-hydroxysterols were found by GC/MS, including clerosterol in relatively high amounts and gorgosterol in low amounts . The latter sterol has not been reported to date in a sponge . Comparisons with Cinachyrella species from other geographical areas show marked differences for both phospholipid fatty acid and sterol compositions.

Comput Biol Chem, 2003 Feb, 27(1), 69 - 76
A HMM-based method to predict the transmembrane regions of beta-barrel membrane proteins; Liu Q et al.; A novel method is developed to model and predict the transmembrane regions of beta-barrel membrane proteins . It is based on a Hidden Markov model (HMM) with architecture obeying those proteins' construction principles . The HMM is trained and tested on a non-redundant set of 11 beta-barrel membrane proteins known to date at atomic resolution with a jack-knife procedure . As a result, the method correctly locates 97% of 172 transmembrane beta-strands . Out of the 11 proteins, the barrel size for ten proteins and the overall topology for seven proteins are correctly predicted . Additionally, it successfully assigns the entire topology for two new beta-barrel membrane proteins that have no significant sequence homology to the 11 proteins . Predicted topology for two candidates for beta-barrel structure of the outer mitochondrial membrane is also presented in the paper.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jun, 35(6), 518 - 21
The phosphorylation of NS protein of wheat rosette stunt virus; Xie BT et al.; The genome of wheat rosette stunt virus (WRSV), a plant rhabdovirus, is a single negative strand RNA . It encodes five viral structural proteins: the glycoprotein (G), the matrix protein (M), the nucleocapsid protein (N), the large protein (L) and the non?structural protein (NS), which was later proved to be a viral structural protein too and existed in a variety of phosphorylation forms in case of vascular stomatitis virus (VSV) . In this paper we demonstrated that NS protein of WRSV, either bound with the viral nucleocapsid or expressed in bacteria could be in vitro phosphorylated in presence of viral nucleocapsid . We concluded that the NS protein of WRSV was a phosphorylated protein and it might exist in both phosphorylated and dephosphorylated forms in virions . Our results excluded the possibility that the NS protein could be autophosphorylated . The L protein, the major component of viral RNA dependent RNA polymerase is associated with the protein kinase for phosphorylation of NS protein.

J Exp Biol, 2003 Jul, 206(Pt 14), 2441 - 57
Food transport in the C . elegans pharynx; Avery L et al.; Pumping of the C . elegans pharynx transports food particles (bacteria) posteriorly . We examined muscle motions to determine how this posterior transport is effected . We find that the motions of the middle section of the pharynx, the anterior isthmus, are delayed relative to the anterior section, the corpus . Simulations in which particles are assumed to move at mean fluid velocity when not captured by the walls of the pharyngeal lumen show that delayed isthmus motions do indeed cause net particle transport; however, the amount is much less than in the real pharynx . We propose that the geometry of the pharyngeal lumen forces particles to the center, where they move faster than mean fluid velocity . When this acceleration is incorporated into the simulation, particles are transported efficiently . The transport mechanism we propose explains past observations that the timing of muscle relaxation is important for effective transport . Our model also makes a prediction, which we confirm, that smaller bacteria are better food sources for C . elegans than large ones.

Eukaryot Cell, 2003 Jun, 2(3), 638 - 45
MFE1, a member of the peroxisomal hydroxyacyl coenzyme A dehydrogenase family, affects fatty acid metabolism necessary for morphogenesis in Dictyostelium spp; Matsuoka S et al.; Beta-oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or D-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2 . We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains . A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA hydratase domain . When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation . Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells . Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages . Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells . Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not . These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development . Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their efficient dispersal in nature.

Xenotransplantation, 2003 Jul, 10(4), 357 - 67
Depletion of anti-Gal antibodies by the intravenous infusion of Gal type 2 and 6 glycoconjugates in baboons; Teranishi K et al.; BACKGROUND: Natural anti-Gal antibodies (NAb) to Gal epitopes play a key role in the rejection of pig cells or organs transplanted into primates . We have investigated the effect on NAb return after extracorporeal immunoadsorption (EIA) of the continuous intravenous (i.v.) infusion of (i) bovine serum albumin conjugated to Gal type 6 oligosaccharides (BSA-Gal) or (ii) a poly l-lysine backbone conjugated to Gal type 2 or 6 oligosaccharides (PLL-Gal) . METHODS: Porcine mobilized peripheral blood progenitor cells (PBPC) obtained by leukapheresis from MHC-inbred miniature swine (n = 9) were infused intravenously (i.v.) into baboons: Group 1 baboons (n = 4) received whole body and thymic irradiation, splenectomy, antithymocyte globulin, cobra venom factor, cyclosporine, mycophenolate mofetil, anti-CD154mAb, porcine hematopoietic growth factors, and EIA before transplantation of high doses (2 to 4 x 1010 cells/kg) of PBPC; Group 2 baboons (n = 3) received the Group 1 regimen plus a continuous i.v . infusion of BSA-Gal for up to 30 days; Group 3 baboons (n = 5) received the Group 1 regimen plus a continuous i.v . infusion of PLL-Gal type 2 (n = 2) or both PLL-Gal types 2 and 6 (n = 3) for up to 30 days . RESULTS: Group 1: NAb returned to pre-PBPC levels within 20-30 days, but there was no induction of antibody to Gal or non-Gal determinants; Group 2: NAb was undetectable or at very low level during BSA-Gal therapy . In one baboon, however, IgG to Gal type 2, but not to type 6, returned during BSA-Gal therapy; Group 3: NAb was undetectable or at very low level during PLL-Gal therapy . In two baboons that received PLL-Gal type 2, NAb to Gal type 6, but not to type 2, returned during PLL-Gal treatment . Two of five baboons, however, developed systemic infection . Four of five baboons died within 14 days; autopsy revealed focal hemorrhagic injury to their hearts, lungs, and small intestines, with histologic abnormalities that varied between animals from hemorrhage and/or thrombosis in some organs (heart, lungs, or intestine) to signs of infections (bacteria in intestine, cytomegalovirus in liver) . CONCLUSIONS: (i) BSA-Gal and PLL-Gal therapy maintained depletion of NAb . (ii) Some heterogeneity in specificity of NAb was identified, indicating that the infusion of a combination of Gal type 2 and 6 glycoconjugates may be required . (iii) The addition of PLL-Gal to the immunosuppressive regimen was associated with a high incidence of morbidity and mortality without a clear histopathologic entity underlying the cause of death.

Mol Plant Microbe Interact, 2003 Jun, 16(6), 508 - 24
A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation; Djordjevic MA et al.; A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics . Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S . meliloti proteins . In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity) . The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity) . The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers . Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total . Proteins with up to seven transmembrane domains were identified in membrane preparations . A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S . meliloti cells in nodules and under stress . Several nodule proteins from the plant host were present in the nodule bacteria preparations . We also identified seven potentially novel proteins not predicted from the DNA sequence . Post-translational modifications such as N-terminal processing could be inferred from the data . The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated . This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Mol Plant Microbe Interact, 2003 Jun, 16(6), 485 - 94
Expression of the baculovirus p35 protein in tobacco affects cell death progression and compromises N gene-mediated disease resistance response to Tobacco mosaic virus; del Pozo O et al.; The p35 protein from baculovirus is a broad-range caspase inhibitor and suppresses programmed cell death in animals . We report here the effects of transgenic expression in tobacco of the p35 protein during the hypersensitive response (HR) . Expression of p35 causes partial inhibition of nonhost HR triggered by bacteria and gene-for-gene HR triggered by virus . Infection of p35-expressing tobacco plants with Tobacco mosaic virus (TMV) disrupts N-mediated disease resistance, causing systemic spreading of the virus within a resistant background . Mutant variants altered in aspartate residues within the loop region of p35 are inefficient substrates for caspases in vitro, and they do not suppress caspase proteolytic activity in animal systems . Tobacco plants expressing these mutant variants of the p35 protein do not show inhibition of HR cell death or enhanced virus systemic movement . Thus, HR inhibition and TMV systemic spreading phenotype in p35-expressing plants correlate with the ability of the p35 protein to suppress caspase activity in animal systems . In addition, a C-terminal truncated variant of p35 is unable to suppress cell death in animals as well as HR cell death in transgenic tobacco . Our results provide evidence for the participation of caspase-like proteases during the HR . In addition, they suggest that timely activation of cell death is necessary for effective TMV containment within the primary infection site.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 263 - 74
Production and energetic use of biogas from energy crops and wastes in Germany; Weiland P; The production of biogas for reducing fossil CO2 emissions is one of the key strategic issues of the German government and has resulted in the development of new process techniques and new technologies for the energetic use of biogas . Progress has been made in cultivating energy crops for biogas production, in using new reactor systems for anaerobic digestion, and in applying more efficient technologies for combined heat and power production . Recently, integration of fuel cells within the anaerobic digestion process was started, and new technologies for biogas upgrading and conversion to hydrogen were tested . This article describes the trends in Germany for achieving more efficient energy production.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 227 - 39
Electron beam pretreatment of sewage sludge before anaerobic digestion; Shin KS et al.; The pretreatment of waste-activated sludge (WAS) by electron beam irradiation was studied in order to improve anaerobic sludge digestion . The irradiation dose of the electron beam was varied from 0.5 to 10 kGy . Batch and continuous-flow stirred tank reactors (CFSTRs) were operated to evaluate the effect of the electron beam pretreatment on anaerobic sludge digestion . Approximately 30-52% of the total chemical oxygen demand (COD) content of the WAS was solubilized within 24 h after electron beam irradiation . A large quantity of soluble COD, protein, and carbohydrates leached out from cell ruptures caused by the electron beam irradiation . Volatile fatty acids production from the irradiated sludge was approx 90% higher than that of the unirradiated sludge . The degradation of irradiated sewage sludge was described by two distinct first-order decay rates (k1 and k2) . Most initial decay reaction accelerated within 10 d, with an average k1 of 0.06/d for sewage sludge irradiated at all dosages . The mean values for the long-term batch first-order decay coefficient (k2) were 0.025/d for irradiated sewage sludge and 0.007/d for unirradiated sludge . Volatile solids removal efficiency of the control reactor fed with unirradiated sewage sludge at a hydraulic retention time (HRT) of 20 d was almost the same as that of the CFSTRs fed with irradiated sludge at an HRT of 10 d . Therefore, disintegration of sewage sludge cells using electron beam pretreatment could reduce the reactor solid retention time by half.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 155 - 66
The reaction front hypothesis in solid-state digestion: estimation of minimum size of viable seed body; Martin DJ et al.; An alternative mechanism for the anaerobic digestion of a bed of solids was recently proposed . A multizoned reaction front might form around a suitable body of seed material, then advance through the bed, within the solid phase . Such a solid-phase mechanism might coexist with liquid-phase digestion but one or the other of these two parallel mechanisms might generally be dominant . It is envisaged that solid-phase digestion would be favored by the presence of viable seed bodies: single particles of a suitable seed material above a minimum size or equivalent aggregates . A key determinant of the feasibility of this mechanism is expected to be the minimum viable size for a seed body (dmin) . This would depend on the thickness of the reaction zones, some of which must initially be accommodated within the seed body . In this article, we present some theoretical estimates of dmin, which indicate a magnitude of 7-700 mm . Such values suggest that solid-phase digestion might be the norm in semi-dry waste digesters . Such digestion might be rare in unseeded landfills but it would appear likely that it could easily be initiated by suitable seeding.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 127 - 38
Anaerobic treatment of animal byproducts from slaughterhouses at laboratory and pilot scale; Edstrom M et al.; Different mixtures of animal byproducts, other slaughterhouse waste (i.e., rumen, stomach and intestinal content), food waste, and liquid manure were codigested at mesophilic conditions (37 degrees C) at laboratory and pilot scale . Animal byproducts, including blood, represent 70-80% of the total biogas potential from waste generated during slaughter of animals . The total biogas potential from waste generated during slaughter is about 1300 MJ/cattle and about 140 MJ/pig . Fed-batch digestion of pasteurized (70 degrees C, 1 h) animal byproducts resulted in a fourfold increase in biogas yield (1.14 L/g of volatile solids {VS}) compared with nonpasteurized animal byproducts (0.31 L/g of VS) . Mixtures with animal byproducts representing 19-38% of the total dry matter were digested in continuous-flow stirred tank reactors at laboratory and pilot scale . Stable processes at organic loading rates (OLRs) exceeding 2.5 g of VS/(L.d) and hydraulic retention times (HRTs) less than 40 d could be obtained with total ammonia nitrogen concentrations (NH4-N + NH3-N) in the range of 4.0-5.0 g/L . After operating one process for more than 1.5 yr at total ammonia nitrogen concentrations >4 g/L, an increase in OLR to 5 g of VS/(L.d) and a decrease in HRT to 22 d was possible without accumulation of volatile fatty acids.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 95 - 105
Codigestion of manure and organic wastes in centralized biogas plants: status and future trends; Angelidaki I et al.; Centralized biogas plants in Denmark codigest mainly manure, together with other organic waste such as industrial organic waste, source sorted household waste, and sewage sludge . Today 22 large-scale centralized biogas plants are in operation in Denmark, and in 2001 they treated approx 1.2 million tons of manure as well as approx 300,000 of organic industrial waste . Besides the centralized biogas plants there are a large number of smaller farm-scale plants . The long-term energy plan objective is a 10-fold increase of the 1998 level of biogas production by the year 2020 . This will help to achieve a target of 12-14% of the national energy consumption being provided by renewable energy by the year 2005 and 33% by the year 2030 . A major part of this increase is expected to come from new centralized biogas plants . The annual potential for biogas production from biomass resources available in Denmark is estimated to be approx 30 Peta Joule (PJ) . Manure comprises about 80% of this potential . Special emphasis has been paid to establishing good sanitation and pathogen reduction of the digested material, to avoid risk of spreading pathogens when applying the digested manure as fertilizer to agricultural soils.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 63 - 76
Dynamic simulation of pH in anaerobic processes; Campos E et al.; With the objective of contributing to the buildup of mathematical tools for anaerobic process simulation, an algorithm for the dynamic simulation of pH was developed . The dynamic simulation of the gaseous phase variables was also considered . The pH algorithm was validated for a watery system, obtaining good agreement between predicted and experimental data . The applied methodology provides a differential equation that allows the inclusion of pH as a state variable of the system that can be easily included in a general mathematical model of anaerobic digestion using matrix notation . This methodology also allows a noticeable decrease in computing time in simulations . A dynamic anaerobic digestion model of complex substrates taken from the literature was completed with the developed algorithms, and it was used to predict the response of an anaerobic reactor against overloading and against the presence of pH-dependent inhibitors with satisfactory results.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 15 - 32
Modeling of anaerobic degradation of solid slaughterhouse waste: inhibition effects of long-chain fatty acids or ammonia; Lokshina LY et al.; The anaerobic bioconversion of solid poultry slaughterhouse wastes was kinetically investigated . The modified version of <METHANE> simulation model was applied for description of experimental data in mesophilic laboratory digester and assays . Additionally, stages of formation and consumption of long chain fatty acids (LCFA) were included in the model . Batch data on volatile solids, ammonium, acetate, butyrate, propionate, LCFA concentrations, pH level, cumulative volume, and methane partial pressure were used for model calibration . As a reference, the model was used to describe digestion of solid sorted household waste . Simulation results showed that an inhibition of polymer hydrolysis by volatile fatty acids and acetogenesis by NH3 or LCFA could be responsible for the complex system dynamics during degradation of lipid- and protein-rich wastes.

Ann N Y Acad Sci, 2003 May, 992, 205 - 17
Stress, leukocyte trafficking, and the augmentation of skin immune function; Dhabhar FS; Delayed type hypersensitivity (DTH) reactions represent cell-mediated immune responses that exert important immunoprotective (resistance to viruses, bacteria, and fungi) or immunopathologic (allergic or autoimmune hypersensitivity) effects . We have used the skin DTH response as an in vivo model to study neuro-endocrine-immune interactions . We hypothesized that just as an acute stress response prepares the cardiovascular and musculoskeletal systems for fight or flight, it may also prepare the immune system for challenges (e.g., wounding) that may be imposed by a stressor (e.g., an aggressor) . Studies showed that acute (2 hours) stress experienced before primary or secondary cutaneous antigen exposure induces significantly enhanced skin DTH . This enhancement involves innate as well as adaptive immune mechanisms . Adrenalectomy eliminates the stress-induced enhancement of DTH . Acute administration of physiological concentrations of corticosterone and/or epinephrine to adrenalectomized animals enhances skin DTH . Compared with those in controls, DTH sites from acutely stressed or hormone-injected animals show significantly greater erythema and induration, numbers of infiltrating leukocytes, and levels of cytokine gene expression . In contrast to acute stress, chronic stress is immunosuppressive . Chronic exposure to corticosterone or acute exposure to dexamethasone significantly suppresses skin DTH . These results suggest that during acute stress, endogenous stress hormones enhance skin immunity by increasing leukocyte trafficking and cytokine gene expression at the site of antigen entry . Elucidation of mechanisms mediating a stress-induced enhancement of skin immune function is important because such immunoenhancement can have protective (wound healing, resistance to infection) or pathological (allergic or autoimmune hypersensitivity) consequences.

Water Sci Technol, 2003, 47(7-8), 327 - 34
Field monitoring and evaluation of innovative solutions for cleaning storm water runoff; Papiri S et al.; Urbanization increases the variety and amount of pollutants transported to receiving waters . Sediment from development and new construction; oil, grease, and toxic chemicals from automobiles; nutrients and pesticides from turf management and gardening; viruses and bacteria from failing septic systems; road salts; and heavy metals are examples of pollutants generated in urban areas . Sediments and solids constitute the largest volume of pollutant loads to receiving waters in urban areas . When runoff enters storm drains, it carries many of these pollutants with it . In older cities, this polluted runoff is often released directly into open waterways without any treatment . Increased pollutant loads can harm fish and wildlife populations, kill native vegetation, foul drinking water supplies, and make recreational areas unsafe . The objective of the study, performed by University of Pavia (Italy), University of Brescia (Italy) and GreenTechTexas International (US), reported herein is to evaluate the use of an innovative stormwater technology (EcoDrain) to reduce pollution due to urban runoff in existing urban areas . The paper describes the methodology and the results achieved with tests conducted in laboratory in Pavia University in Italy and in two pilot areas in Italy and in Australia to investigate the EcoDrain's effectiveness for oil and heavy metals retention and sediment trapping . In the tests performed in a marina near Sydney in Australia a reduction has been achieved in oil and grease concentration higher than 95% and a reduction in metal concentration (particularly Copper, Lead and Zinc) close to 98% . The paper also describes the methodology of the analysis on the absorbing material after its use and the consequent determination of the most efficient and environmentally safe way to dispose of consummated absorbent.

Curr Issues Mol Biol, 2003 Apr, 5(2), 37 - 42
Genomes and evolution; Danchin A; Genomics today involves the study of many genes at a time in order to gain an integrated picture of the cell or organism as a whole . This review considers the architecture and evolution of bacterial genomes . The many facets of large-scale functional investigation in a variety of bacteria and the search to find common rules in their dynamic and structural organization are discussed . Such rules could aid the understanding of common properties and essential differences corresponding to elusive functions, or of still unknown bacterial biotopes.

Histol Histopathol, 2003 Jul, 18(3), 889 - 96
T cell regulation of the immune response to infection in periodontal diseases; Yamazaki K et al.; Although T cells have been implicated in the pathogenesis and are considered to be central both in progression and control of the chronic inflammatory periodontal diseases, the precise contribution of T cells to the regulation of tissue destruction has not been fully elucidated . Current dogma suggests that immunity to infection is controlled by distinct T helper 1 (Th1) and T helper 2 (Th2) subsets of T cells classified on the basis of their cytokine profile . Further, a subset of T cells with immunosuppressive function and cytokine profile distinct from Th1 or Th2 has been described and designated as regulatory T cells . Although these regulatory T cells have been considered to maintain self-tolerance resulting in the suppression of auto-immune responses, recent data suggest that these cells may also play a role in preventing infection-induced immunopathology . In this review, the role of functional and regulatory T cells in chronic inflammatory periodontal diseases will be summarized . This should not only provide an insight into the relationship between the immune response to periodontopathic bacteria and disease but should also highlight areas of development for potentially new therapeutic modalities.

Histol Histopathol, 2003 Jul, 18(3), 787 - 95
Lectin binding patterns in normal canine endometrium and in bitches with pyometra and cystic endometrial hyperplasia; Leitner M et al.; Cystic endometrial hyperplasia (CEH) and pyometra in the bitch are dioestral syndromes, supposed to be caused by hormonal disturbances and changes in endometrial steroid hormone receptor levels . Histologically, the endometria show cystic dilated glands and, if bacteria succeed in invading the uterus, pyometra may develop in the following metoestrus . In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of uterine glands and surface epithelium in healthy dogs and in dogs with CEH and pyometra . Lectin binding is a useful tool to identify glycoconjugates, especially of the glycocalyx, which has essential functions in the endometrium during reproduction . Uterine tissue was obtained from 18 healthy bitches in metoestrus or anoestrus and 18 bitches with a clinical diagnosis of CEH or pyometra . Normal endometria showed cycle-dependent changes in SBA, PNA, HPA and UEA binding during metoestrus and anoestrus . LCA did not show cycle-dependent changes and WGA bound to Golgi regions in the apical parts of surface epithelial cells only in metoestrous . Endometria with inflammatory alterations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, showed a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretions . In dogs with CEH, unaltered glands with generally strong lectin binding to the glycocoalyx and Golgi regions were found adjacent to altered glands . The decrease of lectin binding in pyometra cases is supposed to be a result of glandular exhaustion after cystic hyperplasia . In addition, bacterial adhesion to sugar residues on the uterine surface epithelium might impede lectin binding.

J Infect Dis, 2003 Jun 15, 187 Suppl 2, S356 - 63
Myeloid differentiation factor 88-dependent and -independent pathways in toll-like receptor signaling; Akira S et al.; Toll-like receptors (TLRs) play an essential role in the detection of invading pathogens in the body . Individual TLRs recognize distinct components derived from pathogens, which is followed by cytokine production . The TLR family harbors extracellular leucine-rich repeat domains and a cytoplasmic domain that is homologous to that of the interleukin (IL)-1 receptor (IL-1R) family . After stimulation, TLR recruits IL-1R-associated kinase via adaptor myeloid differentiation factor 88 (MyD88) and induces activation of NF-kappaB and mitogen-activated protein kinases . Cytokine production in response to each TLR ligand is completely abrogated in MyD88-deficient cells, which indicates that MyD88 is an essential shared signaling molecule in the IL-1R/Toll family . The TLR4 signal has an MyD88-independent pathway that is involved in induction of type I interferons (IFNs) and IFN-inducible genes via IFN regulatory factor-3 activation . A recently identified adaptor molecule, Toll-IL receptor domain-containing adaptor protein/MyD88 adaptor-like, may participate in the MyD88-independent pathway.

J Infect Dis, 2003 Jun 15, 187 Suppl 2, S351 - 5
Regulation of receptor-dependent activation of the innate immune response; Ulevitch RJ; In the United States, >750,000 patients annually are thought to be at high risk for developing septic shock, with mortality rates reaching 60% . Thus, huge societal and financial costs are associated with this syndrome . Because of the high incidence and poor prognosis of septic shock, basic research has focused on the innate immune system for >2 decades . The pathophysiology of severe sepsis/shock is exceedingly complex, but there is little doubt that infection often progresses from systemic inflammatory response to severe sepsis and shock . Infection is the primary event in this sequence . There is evidence that the severity of the systemic reaction to infection (severe sepsis) is strongly influenced by the intensity of the inflammatory process at the infection sites . Efforts to understand the molecular mechanisms involved in recognition of bacterial products by members of the Toll-like receptor family are described, as well as some events that occur after receptor ligand binding that lead to new gene activation.

Transplantation, 2003 May 15, 75(9), 1460 - 2
Intestinal decontamination using povidone-iodine compromises small bowel storage quality; Olson DW et al.; BACKGROUND: Povidone-iodine (PI) (Betadine, Purdue Fredrick Co., Norwalk, CT) is used during pancreas transplantation to provide duodenal decontamination without any intestinal complications . METHODS: This study assessed the combined effect of a luminal pretreatment with PI followed by a proven amino acid (AA)-rich preservation solution on small bowel (SB) storage quality . Rat SB was procured using intra-arterial University of Wisconsin solution followed by luminal administration with combinations of AA+/-PI pretreatment as follows: group 1, 20 mL AA solution; group 2, 20 mL PI and 20 mL AA solution; group 3, 100 mL AA solution; and group 4, 20 mL PI and 100 mL AA solution . Cellular energetics, permeability, and histology were analyzed throughout 10-hr cold storage . RESULTS: After 10 hr, adenosine triphosphate levels remained 2.9 to 3.2 micromol/g protein lower in the PI-treated groups than in groups 1 and 3 . Functional parameters were also negatively affected by PI treatment . Mannitol permeability was 203 and 66 nmol/cm2 per hr in groups 2 and 4 versus 46 and 44 nmol/cm2 per hr in groups 1 and 3 . Median grades of histologic injury (Park's) at 10 hr were 3, 5.5, 4, and 5 for groups 1 through 4, respectively . Luminal flushing with PI or AA solution resulted in an equivalent clearance greater than 95% of enteric bacteria present . CONCLUSIONS: hort-duration luminal PI exposure potentiates SB injury and provides no improved intestinal decontamination.

J Clin Microbiol, 2003 Jun, 41(6), 2694 - 6
Stability of repetitive-sequence PCR patterns with respect to culture age and subculture frequency; Kang HP et al.; To examine the stability of repetitive-sequence (rep) PCR profiles, six species of bacteria were subcultured to blood agar plates and DNA was extracted from the cultures after 24, 48, and 72 h of incubation at 35 degrees C . In addition, the same species were subcultured to fresh blood plates daily and DNA was extracted from the cultures after growth of 5, 10, and 15 subcultures, respectively . rep PCR analysis demonstrated that all rep PCR fingerprints from a single species were identical.

Am J Clin Nutr, 2003 Jun, 77(6), 1459 - 65
Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women; Zubik L et al.; BACKGROUND: Test results on the bioavailability of isoflavones in the aglycone or glucoside form in Eastern and Western human subjects are contradictory . OBJECTIVE: The objective was to investigate the bioavailability of the soy isoflavones daidzein and genistein in American women with typical American dietary habits after ingestion of the aglycone or glucoside form of isoflavones . DESIGN: Fifteen American women aged 46 +/- 6 y participated in a randomized, double-blind study . Blood samples were collected 0, 1, 2, 4, 8, 12, 24, and 48 h after consumption of aglycone or glucoside tablets with breakfast . The plasma curves for daidzein, genistein, and equol were constructed and the postprandial maximum concentration (C(max)), time to the maximum concentration (t(max)), and area under the curve (AUC) were determined . RESULTS: Isoflavone concentrations peaked early (1-2 h) in plasma and peaked again at 4-8 h . Mean C(max), t(max), and AUC values for genistein were not significantly different after ingestion of aglycone or glucoside . However, C(max) and AUC values, but not t(max), were significantly higher for daidzein after aglycone ingestion, which was partly due to its higher content in the aglycone tablets . Equol appeared after 4 h and remained elevated after 48 h . Despite a higher content of daidzein in the aglycone tablets, the AUC for equol was significantly higher after ingestion of the glucoside tablets, probably because of the metabolic action of intestinal bacteria during the long intestinal transit time of glucoside . CONCLUSION: The apparent bioavailability of genistein and daidzein is not different when consumed as either aglycone or glucoside by American women.

Curr Allergy Asthma Rep, 2003 Jul, 3(4), 352 - 7
Epithelial cells in ocular allergy; Irkec M et al.; Conjunctival epithelial cells do not act only as mechanical barriers, preventing the entry of particles, bacteria, viruses, and noxious substances into the eye but they are also active participants in the regulation of allergic inflammation via expressing adhesion/effector molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, human leukocyte antigen-DR, CD40/CD40L, Fas/Fas ligand) on their surfaces and releasing numerous proinflammatory cytokines, such as eotaxin, regulated on activation normal T-cell expressed and released (RANTES), macrophage inflammatory protein-1, interleukin (IL)-8, IL-6, and tumor necrosis factor-a, which are necessary for the proliferation, differentiation, activation, and chemotaxis of various inflammatory cells into the conjunctiva . Histamine, released from the conjunctival mast cells, might stimulate the synthesis of proinflammatory molecules such as IL-6 and IL-8 by the epithelial cells through the receptors that couple to inositol phosphate generation and, therefore, amplify the allergic response . The relationship between the epithelium and allergy should be considered in detail in future studies aiming at an effective control and treatment of all forms of allergic conjunctivitis.

Compend Contin Educ Dent, 2002 Jul, 23(7 Suppl 1), 7 - 10
Impact of the sonicare toothbrush on plaque and gingivitis; Kugel G et al.; The sonicare toothbrush has been shown to be 40% more effective in removing plaque than a manual toothbrush, 82% better in removing plaque from interproximal areas, and to reverse gingivitis . In vitro studies have examined the ability of the sonicare toothbrush to remove plaque bacteria beyond the reach of the bristles . The sonicare toothbrush has also been shown to produce significantly less dentin wear in vitro than another leading power toothbrush . This article reviews both in vivo and in vitro studies that illustrate the efficacy of the sonicare toothbrush in improving oral health.

Compend Contin Educ Dent, 2002 May, 23(5 Suppl), 11 - 4
The etiology and pathogenesis of periodontitis; Page RC; Major advances have been made in our basic understanding of periodontal disease during the last decade . Pathways whereby bacteria destroy the periodontal connective tissues and alveolar bone have been elucidated . The manner in which various risk factors alter these pathways to enhance risk and amplify the onset and progression of disease is becoming clearer . Furthermore, a strong link between periodontitis and potentially fatal systemic diseases and conditions has been demonstrated . These advances in knowledge have led to the development of new approaches to treatment that enhance our capacity to prevent and control oral infections and to decrease potential systemic disease consequences.

Compend Contin Educ Dent, 2002 May, 23(5 Suppl), 3 - 10
A century of progress in understanding periodontal disease; Williams RC; Throughout the 20th century, investigators and clinicians sought to discover the causes and trace the natural history of periodontal disease . Noteworthy progress has been made on several fronts . It was once believed that oral hygiene and age accounted predominantly for variances in the prevalence and severity of periodontal disease; now, a number of innate, acquired, and environmental risk factors have been identified . Light has been shed on the roles in periodontal disease pathogenesis of both specific bacteria and bacterial complexes and host immunoinflammatory responses . Insight into periodontal wound healing has fostered promising approaches to promoting regeneration of damaged periodontal structures . Finally, although theories of "focal infection" as a primary cause of systemic disease have been discredited, recent studies have confirmed the existence of an intimate connection between oral and systemic health . The progress made in understanding the nature of periodontal disease has been complemented by equally noteworthy therapeutic advances . The coupling of surgical and medical approaches to treatment ushers in a new era in the management of periodontal disease.

J Am Acad Dermatol, 2003 Jun, 48(6), 878 - 81
Cutaneous lesions of secondary syphilis are highly angiogenic; Macaron NC et al.; BACKGROUND: The role of angiogenesis in infectious processes is poorly studied . Some viruses have been linked to angiogenesis, but the role of bacteria and protozoa in inducing angiogenesis in chronic infections is poorly understood . OBJECTIVES: We examined the role of angiogenesis in syphilis, a common and often difficult-to-treat infectious disease, especially in the setting of HIV/AIDS . METHOD: Microvessel counts were performed on 27 paraffin-fixed sections of secondary syphilis by staining with monoclonal antibodies against CD31 . In addition, immunohistochemistry was performed using antibodies against vascular endothelial growth factor (VEGF) to determine whether increased angiogenesis may be mediated, in part, through increased production of VEGF . RESULTS: The CD31 mean microvessel count in secondary syphilis sections was significantly higher than in normal control sections . VEGF intensity appeared increased in the patients with secondary syphilis . CONCLUSIONS: Infection with Treponema pallidum results in increased angiogenesis in secondary syphilis . The mechanism for increased angiogenesis may involve elaboration of angiogenic cytokines, such as VEGF and epidermal growth factor.

Afr Health Sci, 2002 Aug, 2(2), 52 - 5
Comparison of immunohistochemical and modified Giemsa stains for demonstration of Helicobacter pylori infection in an African population; Wabinga HR; BACKGROUND: Modified Giemsa staining has been favoured by many researchers because it is easy to perform but, like many other stains, demonstration of the bacteria depends on its morphology . It has been arged in some research circles that some of the organisms in the gastric mucosa may not be true H.pylori . Immunohistochemical techniques have been developed and make use of anti H.pylori antibody, which reacts, with somatic antigens of the whole bacteria and have been found to correlate well with the presence of the bacteria . OBJECTIVE: To ascertain the efficacy of modified Giemsa stain in an African setting where H.Pylori seems quite prevalent . STUDY DESIGN: A laboratory-based study of two diagnostic tests in which modified Giema stain was compared with immunohistochemistry . METHODS: A total of 48 consecutive autopsy cases with no upper gastro intestinal diseases had their gastric mucosa stained for demonstration of H.pylori using both modified Giemsa and immunohisto chemical staining techniques . RESULTS: Twenty-seven cases of H.pylori were demonstrated by both techniques and 14 cases were not identified by the two staining methods . In 2 cases immunostain could not demonstrate the bacteria but they were identified with modified Giemsa stain while in 5 cases the bacteria were identified by immunostain but not with modified Giemsa stain . The sensitivity of modified Giemsa stain was 85% (CI 66.5-98.8) while the specificity was 89% (CI 60.4 - 97.8) . The positive predictive value of modified Giemsa stain was 93% CI 75 - 98.8%) while the negative predictive value was 74% (CI 48.6 - 89.9) . The kappa statistic comparing the 2 stains was 0.69 (p value 0.00001) giving a good agreement between the two tests . CONCLUSION: With the above results the modified Giemsa stain, which is readily available in most African laboratories, is recommenced for diagnosis of H.pylori, a prevalent infection in Africa.

Virology, 2003 May 25, 310(1), 157 - 62
The mature reverse transcriptase molecules in virions of mouse mammary tumor virus possess protease-derived sequences; Entin-Meer M et al.; Our efforts to express in bacteria the enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV) have shown that the RT is active only after adding 27 amino acid residues, which are derived from the end of the pro gene, to the amino-terminus of the RT (Biochem, J . (1998) 329, 579-587) . In the present study we have tested whether the mature RT found in virions is also fused to protease-derived sequences . To this end, we have analyzed the RT molecules in virions of MMTV by using two antisera directed against peptides, derived from either the carboxyl-terminus of MMTV protease or the middle of MMTV RT . The data suggest that the mature RT, located in virions, contains at its amino-terminus sequences from the carboxyl-terminus of the protease protein . This finding supports previous suggestions that MMTV RT is a transframe protein (derived from both pro and pol reading frames of MMTV) and that amino acid residues located at the carboxyl-terminus of the protease have a dual usage as integral parts of both the protease and the RT enzymes.

FEBS Lett, 2003 Jun 12, 545(1), 18 - 24
Proton translocation by transhydrogenase; Jackson JB; Transhydrogenase, in animal mitochondria and bacteria, couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane . Within the protein, the redox reaction occurs at some distance from the proton translocation pathway and coupling is achieved through conformational changes . In an 'open' conformation of transhydrogenase, in which substrate nucleotides bind and product nucleotides dissociate, the dihydronicotinamide and nicotinamide rings are held apart to block hydride transfer; in an 'occluded' conformation, they are moved into apposition to permit the redox chemistry . In the two monomers of transhydrogenase, there is a reciprocating, out-of-phase alternation of these conformations during turnover.

Bioorg Med Chem, 2003 Jul 3, 11(13), 2835 - 41
Synthesis and biological evaluation of novel turn-modified gramicidin S analogues; Grotenbreg GM et al.; The synthesis of novel gramicidin S analogues having additional functionalities in the turn region by employing a biomimetic approach is described . The preservation of beta-sheet character in all analogues was established by NMR and the biological activity was evaluated.

Biochim Biophys Acta, 2003 Jun 17, 1641(1), 1 - 12
Chaperones and folding of MHC class I molecules in the endoplasmic reticulum; Paulsson K et al.; In this review we discuss the influence of chaperones on the general phenomena of folding as well as on the specific folding of an individual protein, MHC class I . MHC class I maturation is a highly sophisticated process in which the folding machinery of the endoplasmic reticulum (ER) is heavily involved . Understanding the MHC class I maturation per se is important since peptides loaded onto MHC class I molecules are the base for antigen presentation generating immune responses against virus, intracellular bacteria as well as tumours . This review discusses the early stages of MHC class I maturation regarding BiP and calnexin association, and differences in MHC class I heavy chain (HC) interaction with calnexin and calreticulin are highlighted . Late stage MHC class I maturation with focus on the dedicated chaperone tapasin is also discussed.

Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 98 - 103
New functions of lactoferrin and beta-casein in mammalian milk as cysteine protease inhibitors; Ohashi A et al.; We found new inhibitory function of lactoferrin and beta-casein in milk against cysteine proteases using reverse zymography . The inhibition of cathepsin L by lactoferrin was strongest and the inhibition kinetics were of a non-competitive type . Heat denatured lactoferrin lost the inhibitory activity completely, therefore the tertiary structure is essential to show the inhibition . Native lactoferrin was not degraded by papain during the assay condition . The intramolecular peptide, Y(679)-K(695), of lactoferrin is an active domain and the synthesized peptide inhibited cysteine proteases . The Y(679)-K(695) peptide showed 90% homology with the sequences of a common active site of cystatin family . beta-Casein and the active domain, synthesized L(133)-Q(151), peptide inhibited cysteine proteases . Lactoferrin and beta-casein in milk might play a role in antiseptic and antiinfectious functions due to cysteine protease inhibition of bacteria and viruses.

Fish Shellfish Immunol, 2003 Jul, 15(1), 1 - 11
In vitro effect of chitin particles on the innate cellular immune system of gilthead seabream (Sparus aurata L.); Cuesta A et al.; The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied . Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM . Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones . Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated . Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test . Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected . As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner . Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected . To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.

Mar Pollut Bull, 2003, 47(1-6), 78 - 84
Study of the effect of water-soluble fractions of heavy-oil on coastal marine organisms using enclosed ecosystems, mesocosms; Ohwada K et al.; Mesocosm facilities composed of 4 experimental and 2 reservoir tanks (1.5 m in diameter, 3.0 m in depth and 5 tons in capacity) made of FRP plastics, were constructed in the concrete fish rearing pond in the Fisheries Laboratory, The University of Tokyo . The water-soluble fraction of Rank A heavy residual oil was formed by mixing 500 g of the oil with 10 l of seawater, which was introduced to the 5000 l-capacity tanks . Experimental Run 4 was conducted from May 31 to June 7, 2000 . Oil concentrations in the tanks were 4.5 microg/l called LOW, and 13.5 microg/l, called HIGH tank . Bacterial growth rates very quickly accelerated in the HIGH tank just after the loading of oil which corresponded with a high increase of bacterial cells in the same tank after 2 days . Later, bacterial numbers in HIGH tank rapidly decreased, corresponding with the rapid increase of heterotrophic nano-flagellates and virus numbers on the same day . Sediment traps were deployed at the bottom of the experimental tanks, and were periodically retrieved . These samples were observed both under light microscope and epi-fluorescent microscope with UV-excitation . It was observed that the main components of the vertical flux were amorphous suspended matter, mostly originating from dead phytoplankton and living diatoms . It was further observed from the pictures that vertical transport of oil emulsions were probably conducted after adsorption to amorphous suspended matter and living diatoms, and were settling in the sediment traps at the bottom of the tanks . This means that the main force which drives the soluble fraction of oil into bottom sediment would be vertical flux of such amorphous suspended particles and phytoplankton . Further incubation of the samples revealed that the oil emulsions were degraded by the activity of autochtonous bacteria in the sediment in aerobic condition.

Mar Pollut Bull, 2003, 47(1-6), 5 - 9
Assay of phosphatase activity and ATP biomass in tideland sediments and classification of the intertidal area using chemical values; Nakamura K et al.; Detailed studies of the phosphatase activity in the tidelands were examined including the localization of the phosphatase activities and the spatial distributions of the activity in the tidelands around Hiroshima Bay, the Aki-Bingo Open Sea and Isahaya Bay of Kyushu on the western side of Japan . The results indicate that the phosphatase activity of the sediment fraction accounts for 98% of the tideland activity . Furthermore, we classified 19 tidelands into four groups by a cluster analysis using two parameters: phosphatase activities and ATP (adenosine 5(')-triphosphate) biomass . The first group is a member of the tidelands located in areas remote from the cities . They have a low phosphatase activity and low ATP biomass and the seawater in the tideland is very clean . The second is a group with a low phosphatase activity and high ATP biomass . In the tidelands belonging to this group, large numbers of benthos and shellfish can be observed . The third group has a high phosphatase activity and a relatively low ATP biomass . The estuary tidelands located near Hiroshima City belong to the third group . The last one is tidelands with a high phosphatase activity and high ATP biomass . In this group, the ratio of the phosphatase activity to ATP biomass is relatively low . In the summer, we observed deterioration in the water quality and accumulation of sludge on the sea bottom in the tideland of the last group.Moreover, we employed a multiple regression analysis and proposed an equation to obtain the group number of the cluster analysis using the assayed values of the phosphatase activity and the ATP biomass.

Cell Mol Life Sci, 2003 Apr, 60(4), 648 - 62
Molecular adaptations to cold in psychrophilic enzymes; Feller G; Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earth's biosphere . To cope with the reduction of chemical reaction rates induced by low temperatures, these organisms synthesize enzymes characterized by a high catalytic activity at low temperatures associated, however, with low thermal stability . Thanks to recent advances provided by X-ray crystallography, protein engineering and biophysical studies, we are beginning to understand the molecular adaptations responsible for these properties which appear to be relatively diverse . The emerging picture suggests that psychrophilic enzymes utilize an improved flexibility of the structures involved in the catalytic cycle, whereas other protein regions if not implicated in catalysis may or may not be subjected to genetic drift.

Indian J Pathol Microbiol, 2002 Jul, 45(3), 299 - 302
A study of keratomycosis: our experience; Kumari N et al.; The aim of the present study was to determine the etiological agents, predisposing factors and important diagnostic tests in keratomycosis in Patna . During period of 2 years, 204 clinically suspected cases of keratomycosis were studied by microscopy (10% KOH smear preparation and Gram's staining) and culture (for bacteria as well as fungus) . Out of 204 clinically suspected cases, 76 cases (37.23%) were reported positive for fungus by microscopy and culture . 77% cases were between the age group of 21 to 50 years . Males were more than double the number of females affected . 82% patients were farmers by occupation . 81.58% cases gave a definite history of antecedent corneal trauma . 6.58% cases were known diabetics . Peak incidence was recorded in the months of September-October of the years . Single fungal isolates were present in 63 cases (31%) and multiple were there in 3 cases (1.47%) . Mixed isolates of bacteria and fungi were there in 4.90% cases . The predominant isolate was Aspergillus species--42 cases (52.26%) followed by Candida--7 cases (9.21%), Penicillium--6 cases (7.89%) and Fusarium-6 cases (7.89%) . Ten (13.15%) of fungal isolates remained unidentified . Therefore mycotic keratitis should be suspected in every patient with a corneal lesion and should be ruled out promptly to save sight and before commencing steroids and antibiotics.

Rinsho Shinkeigaku, 2002 Nov, 42(11), 1102 - 4
{Medical approach to intraparenchymal spinal cord disorders}; Mukai E; Diagnosis depends on the clinical manifestations, blood or cerebrospinal fluid study and MRI findings . Acute and subacute intraparenchymal spinal cord disorders are due to vascular disorders or myelitis . Spinal cord infarction is associated with dissecting aortic aneurysm, surgical clipping of aortic aneurysms, aortic atherosclerosis or hypotension from any cause . Hematomyelia results from trauma, vascular malformations, vasculitis, or a coagulation disorder . Acute infectious myelopathies result from direct invasion of the spinal cord by bacteria, parasite, or virus . The cause of acute or subacute inflammatory disease include multiple sclerosis, Devic disease, acute disseminated encephalomyelitis, SLE, or sarciodosis . Sarcoidosis sometimes requires differential diagnosis with cord tumor . Chronic intraparenchymal spinal cord disorders are due to syringomyelia, familial spastic paraplegia, HTLV-1 associated myelopathy, adrenomyeloneuropathy, and vascular malformations . HTLV-1 associated myelopathy present with progressive spastic paraplegia with bladder disturbance and has antibodies to HTLV-1 in the cerebrospinal fluid and serum . Diagnosis of adrenomyeloneuropathy is made by demonstration of elevated levels of very long chain fatty acids in plasma . Vascular malformations are important lesions because they present a treatable cause of progressive myelopathy.

Rinsho Shinkeigaku, 2002 Nov, 42(11), 1091 - 4
{Dystroglycan linkage and muscular dystrophy}; Shimizu T; Dystroglycan is a key complex between basal lamina laminin, extracellularly and membrano-cytoskeleton, intracellularly . The damage of this linkage is turned out to cause muscular dystrophies . Dystroglycan knockout is lethal . Dystroglycan-associated intracellular proteins such as dystrophin, dystrobrevin, sarcoglycans, plectin and caveolin-3 are responsible for causing severe (Duchenne type) and moderate forms (Becker, LGMDs) . Laminin, dystroglycan-binding extracellular protein, is deficient in the most severe form of congenital muscular dystrophy with normal intelligence and eye . Recently, a remarkable progress is made in most severe forms of congenital muscular dystrophy with anomalies of brain and eye such as Fukuyama type (Japan) and muscle-eye-brain disease (Finland) . The gene product for Fukuyama type, fukutin, belongs to a family of glycosylation enzymes in bacteria and yeast . Since alpha-dystroglycan contains 14-15 o-glycans, ser/thr-mannose 2-1 GlcNAc 4-1 Gal 3-2 Sial in the middle third mucin-domain and the sial-o-glycan is essential for laminin-binding, and since alpha-dystroglycan is defective in Fukuyama type sarcolemma with anti both sugar moiety- and peptide-antidodies, defective fukutin causes incomplete o-glycosylation of alpha-dystroglycan . In '02, it is clarified that a glycosylation enzyme, POMGnT1 which modifies GlcNAc onto ser/thr-mannose, is defective in 6 MEB patients . The loss of the enzyme activity is turned out to lose alpha-dystroglycan from sarcolemma of MEB . These data strongly suggests that o-glycosylation defect of alpha-dystroglycan causes the most severe congenital muscular dystrophy such as Fukuyama type, MEB and Walker Warburg syndrome.

J Biol Chem, 2003 Sep 19, 278(38), 36373 - 9 Epub 2003 Jun 03.
hFis1, a novel component of the mammalian mitochondrial fission machinery; James DI et al.; The balance between the fission and fusion mechanisms regulate the morphology of mitochondria . In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division . hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission . This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus . Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis . Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation . Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.

Parasitol Res, 2003 Jun, 90(3), 249 - 56 Epub 2003 Mar 19.
Electron microscopic identification of the intestinal protozoan flagellates of the xylophagous cockroach Parasphaeria boleiriana from Brazil; Brugerolle G et al.; Flagellate protozoa of the hindgut of the xylophagous blattid Parasphaeria boleiriana were examined by light and electron microscopy . This species harbours two oxymonad species of the genera Monocercomonoides and Polymastix, the latter bearing Fusiformis bacteria on its surface . A diplomonad was present and has features of the genus Hexamita rather than Spironucleus . In addition, two trichomonads of the genera Monocercomonas and Tetratrichomastix were identified . A precise comparison with species of blattids and other insects was difficult because most of these flagellates have been described only by light microscopy after cell staining and there are few electron microscope studies and no molecular studies . None of the flagellates contained wood fragments in their food vacuoles and so evidently do not participate in the digestion of wood or cellulose.

Rev Physiol Biochem Pharmacol, 2003, 147, 47 - 74 Epub 2003 Feb 21.
Cytochrome c oxidase--structure, function, and physiology of a redox-driven molecular machine; Richter OM et al.; Cytochome c oxidase is the terminal member of the electron transport chains of mitochondria and many bacteria . Providing an efficient mechanism for dioxygen reduction on the one hand, it also acts as a redox-linked proton pump, coupling the free energy of water formation to the generation of a transmembrane electrochemical gradient to eventually drive ATP synthesis . The overall complexity of the mitochondrial enzyme is also reflected by its subunit structure and assembly pathway, whereas the diversity of the bacterial enzymes has fostered the notion of a large family of heme-copper terminal oxidases . Moreover, the successful elucidation of 3-D structures for both the mitochondrial and several bacterial oxidases has greatly helped in designing mutagenesis approaches to study functional aspects in these enzymes.

J Biol Chem, 2003 Aug 8, 278(32), 29760 - 8 Epub 2003 Jun 02.
Gts1p activates SNF1-dependent derepression of HSP104 and TPS1 in the stationary phase of yeast growth; Yaguchi S et al.; We previously reported that the GTS1 product, Gts1p, plays an important role in the regulation of heat tolerance of yeast under glucose-limited conditions in either batch or continuous culture . Here we show that heat tolerance was decreased in GTS1-deleted and increased in GTS1-overexpressing cells under glucose-derepressed conditions during the batch culture and that the disruption of SNF1, a transcriptional activator of glucose-repressible genes, diminished this effect of GTS1 . Intracellular levels of Hsp104 and trehalose, which were reportedly required for the acquisition of heat tolerance in the stationary phase of cell growth, were affected in both GTS1 mutants roughly in proportion to the gene dosage of GTS1, whereas those of other Hsps were less affected . The mRNA levels of genes for Hsp104 and trehalose-6-phosphate synthase 1 changed as a function of GTS1 gene dosage . The Q-rich domain of Gts1p fused with the DNA-binding domain of LexA activated the transcription of the reporter gene LacZ, and Gts1p lacking the Q-rich domain lost the activation activity of HSP104 and TPS1 . Furthermore, Gts1p bound to subunits of Snf1 kinase, whereas it did not bind to DNA . Therefore, we suggested that GTS1 increases heat tolerance by mainly activating Snf1 kinase-dependent derepression of HSP104 and TPS1 in the stationary phase of yeast growth.

Virus Res, 2003 Jun, 93(2), 151 - 8
Spectroscopic characterization of two peptides derived from the stem of rabies virus glycoprotein; Maillard A et al.; Rabies virus glycoprotein (G) is a trimeric type I transmembrane glycoprotein that mediates both receptor recognition and low pH-induced membrane fusion . Electron microscopy has indicated that the ectodomain of protein G is made of a globular head and a stem . In order to characterize the putative stem region at the molecular level, we designed two peptides, P(S) and P(L), which were produced as GST fusion proteins in bacteria . Peptide P(S) extends from amino acid (aa) 374 to aa 428 whereas peptide P(L) extends from aa 368 down to the end of the ectodomain of G (aa 439) . Their secondary and quaternary structures have been studied with spectroscopic and biophysical methods . We show that these isolated peptides are monomeric and poorly structured in aqueous solution . However, circular dichroism (CD) in presence of 2,2,2-trifluoroethanol and NMR data indicate that this region may adopt a alpha-helical conformation in the complete glycoprotein.

Int J Parasitol, 2003 May, 33(5-6), 469 - 78
Recent advances in veterinary vaccine adjuvants; Singh M et al.; Next generation veterinary vaccines are going to mainly comprise of either subunit or inactivated bacteria/viruses . These vaccines would require optimal adjuvants and delivery systems to accord long-term protection from infectious diseases in animals . There is an urgent need for the development of new and improved veterinary and human vaccine adjuvants . Adjuvants can be broadly divided into two classes, based on their principal mechanisms of action: vaccine delivery systems and 'immunostimulatory adjuvants' . Vaccine delivery systems are generally particulate e.g . emulsions, microparticles, ISCOMS and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC) . In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns, e.g . LPS, MPL and CpG DNA, which activate cells of the innate immune system . Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants might enhance this process in animals and humans alike.

Am J Surg, 2003 Jun, 185(6), 550 - 5
Interleukin-18 concentration in the peritoneal fluid correlates with the severity of peritonitis; Ikuta S et al.; BACKGROUND: Interleukin (IL)-18 is a novel cytokine that has recently been characterized as an inducer of interferon-gamma (IFN-gamma) . The aim of this study was to investigate the clinical significance of peritoneal IL-18 concentrations in patients with peritonitis . METHODS: We measured IL-18, IFN-gamma, and IL-10 concentrations in the peritoneal fluid of 28 patients undergoing laparotomy for peritonitis . Correlations between the peritoneal cytokine concentrations and the severity of illness determined by systemic inflammatory response syndrome (SIRS) criteria, Acute Physiological and Chronic Health Evaluation II (APACHE II) score, peritoneal fluid bacterial culture results, subsequent development of organ failure, and length of hospital stay were assessed . RESULTS: Interleukin-18 concentration was significantly increased in patients who developed SIRS, in those with culture-positive peritonitis, and in those who developed organ failure, as compared with the other patients . Interleukin-10 concentration, which was also significantly increased in patients with culture-positive peritonitis, showed a close correlation with IL-18 concentration . Although a weak correlation was observed between IL-18 and IFN-gamma concentrations, IFN-gamma concentrations did not show any association with patients' clinical parameters . However, the IFN-gamma/IL-18 ratio was significantly lower in patients with an APACHE II >/=10, and in those with culture-positive peritonitis, as compared with the other patients . BACKGROUND: Peritoneal IL-18 concentration increased in response to intraperitoneal bacterial infection and seemed to reflect the severity of peritonitis . However, the capacity of IL-18 to produce IFN-gamma may be altered depending on the severity of peritonitis.

Trends Microbiol, 2003 May, 11(5), 220 - 3
Life without dihydrofolate reductase FolA; Myllykallio H et al.; Reduced folate derivatives participate in numerous reactions of bacterial intermediary metabolism . Consequently, the well-characterized enzyme implicated in the formation of tetrahydrofolate--dihydrofolate reductase FolA--was considered to be essential for bacterial growth . However, comparative genomics has revealed several bacterial genome sequences that appear to lack the folA gene . Here, we provide in silico evidence indicating that folA-lacking bacteria use a recently discovered class of flavin-dependent thymidylate synthases for deoxythymidine-5'-monophosphate synthesis, and propose that many bacteria must contain uncharacterized sources for reduced folate molecules that are still waiting to be discovered.

Trends Microbiol, 2003 May, 11(5), 215 - 9
What is the nature of the replicative niche of a stealthy bug named Brucella?
Kohler S, Michaux-Charachon S, Porte F, Ramuz M, Liautard JP.
Brucella spp . are facultatively intracellular bacteria that persist and multiply in the macrophages of their mammalian hosts . The so-called phagosome to which they have adapted is their natural living niche . Characterization of this niche would facilitate an understanding of the true relationship between the host cell and the intracellular bacteria . This Opinion analyses and discusses the characteristic properties and genesis of this vacuole during phagocytosis as deduced from the virulence factors necessary for intracellular multiplication of the pathogen . We conclude that the replicative niche of Brucella spp.--the 'brucellosome'--differs from all other cellular organelles, and that it isolates the pathogen from certain cytoplasmic nutrients . Adaptation to the stress conditions encountered and the use of anaerobic respiration enable brucellae to replicate in the compartment they create.

Lancet Infect Dis, 2003 Jun, 3(6), 338 - 48
Marine natural products and their potential applications as anti-infective agents; Donia M et al.; The oceans are a unique resource that provide a diverse array of natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria . As infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases . Many marine natural products have successfully advanced to the late stages of clinical trials, including dolastatin 10, ecteinascidin-743, kahalalide F, and aplidine, and a growing number of candidates have been selected as promising leads for extended preclinical assessment . Although many marine-product clinical trials are for cancer chemotherapy, drug resistance, emerging infectious diseases, and the threat of bioterrorism have all contributed to the interest in assessing natural ocean products in the treatment of infectious organisms . In this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent in-vitro activity against infectious and parasitic diseases.

Cell Microbiol, 2003 Jun, 5(6), 373 - 83
Show me the substrates: modulation of host cell function by type IV secretion systems; Nagai H et al.; Evidence for the involvement of type IV protein secretion systems in bacterial virulence is accumulating . Many of the substrate proteins secreted by type IV systems either hijack or interfere with specific host cell pathways . These substrates can be injected directly into host cells via the type IV apparatus or are secreted by the type IV machinery in a state that allows them to gain access to cellular targets without the further assistance of the type IV system . Arguably, the protein substrates of most type IV secretion systems remain undiscovered . Here, we review the activities of known type IV substrates and discuss the putative roles of unidentified substrates.

Clin Exp Immunol, 2003 Jun, 132(3), 393 - 400
Absence of CD4+CD25+ regulatory T cells is associated with a loss of regulation leading to increased pathology in Helicobacter pylori-infected mice; Raghavan S et al.; Helicobacter pylori induces symptomatic chronic gastritis in a subpopulation of infected individuals . The mechanism(s) determining the development and severity of pathology leading to symptoms are not fully understood . In a mouse model of H . pylori infection we analysed the influence of immunoregulatory CD4+CD25+ T cells on H . pylori colonization and gastritis . Athymic C57BL/6 nu/nu mice were reconstituted with (a) lymph node (LN) cells (b) LN cells depleted of CD25+ T cells (CD25(-) LN) or (c) not reconstituted at all . Mice were then infected orally with 3 x 10(8)H . pylori SS1 bacteria . At 2 and 6 weeks after the inoculation there was a significant (P < 0.001) reduction in H . pylori colonization in athymic mice transferred with CD25(-) LN cells compared to mice transferred with LN cells . Colonization was still reduced at 12 weeks after inoculation . Mice transferred with CD25(-) LN cells showed an earlier onset and increased severity of gastritis as compared to mice receiving LN cells . Splenic cells isolated from mice receiving CD25(-) LN cells produced the highest level of IFN-gamma on stimulation with H . pylori antigens in vitro, had a higher H . pylori-specific DTH response and increased infiltration of CD4+ T cells and macrophages in the gastric mucosa . Athymic mice not transferred with T cells had persistent high H . pylori colonization and displayed a normal gastric epithelium without inflammatory cells . In conclusion, CD4+CD25+ cells reduce immunopathology in H . pylori infection, possibly by reducing the activation of IFN-gamma producing CD4+ T cells, even at the expense of a higher H . pylori load in the gastric mucosa.

APMIS, 2003 Apr, 111(4), 474 - 6
Comamonas testosteroni meningitis in a patient with recurrent cholesteatoma; Arda B et al.; Comamonas testosteroni, a lesser-known member of the genus, has shown little apparent capacity for causing infections in humans . We here present a case of purulent meningitis due to C . testosteroni, which occurred in a patient who had recurrent cholesteatoma . Ceftriaxone treatment was not effective in this patient even though in vitro the bacteria were susceptible to the drug . The patient responded well to meropenem therapy.

Chaos, 1995 Mar, 5(1), 155 - 161
A basic mathematical model of the immune response; Mayer H et al.; Interaction of the immune system with a target population of, e.g., bacteria, viruses, antigens, or tumor cells must be considered as a dynamic process . We describe this process by a system of two ordinary differential equations . Although the model is strongly idealized it demonstrates how the combination of a few proposed nonlinear interaction rules between the immune system and its targets are able to generate a considerable variety of different kinds of immune responses, many of which are observed both experimentally and clinically . In particular, solutions of the model equations correspond to states described by immunologists as "virgin state," "immune state" and "state of tolerance." The model successfully replicates the so-called primary and secondary response . Moreover, it predicts the existence of a threshold level for the amount of pathogen germs or of transplanted tumor cells below which the host is able to eliminate the infectious organism or to reject the tumor graft . We also find a long time coexistence of targets and immune competent cells including damped and undamped oscillations of both . Plausibly the model explains that if the number of transformed cells or pathogens exeeds definable values (poor antigenicity, high reproduction rate) the immune system fails to keep the disease under control . On the other hand, the model predicts apparently paradoxical situations including an increased chance of target survival despite enhanced immune activity or therapeutically achieved target reduction . A further obviously paradoxical behavior consists of a positive effect for the patient up to a complete cure by adding an additional target challenge where the benefit of the additional targets depends strongly on the time point and on their amount . Under periodically pulsed stimulation the model may show a chaotic time behavior of both target growth and immune response . (c) 1995 American Institute of Physics.

J Pediatr Surg, 2003 Jun, 38(6), 840 - 3
Gene alteration of intestinal intraepithelial lymphocytes with administration of total parenteral nutrition; Wildhaber BE et al.; BACKGROUND: Total parenteral nutrition (TPN) is associated with sepsis and loss of immune reactivity . The authors previously have shown that changes in the intestinal mucosal immune system--ie, intraepithelial lymphocytes (IEL)--lead to a loss of epithelial barrier function . This may be a mechanism by which bacteria and toxins endanger individuals receiving TPN . To identify altered IEL gene expression during TPN administration, microarray assays were used . METHODS: Mice received oral feeding (control) or TPN for 7 days . Small bowel IEL were separated and retained, RNA purified, and microarray assays performed (Affymetrix system, 12,491 genes) . Results were expressed as quantile-normalized trimmed-means . Significance equals a greater than 2-fold change (TPN v control), P <.01 (t test) or greater than 3-fold, P <.05 . RESULTS: In the TPN group 88, IEL genes were significantly up regulated and 114 downregulated (v control) . Of these genes, 4 were identified to have highest degree of upregulation (FK506-binding protein 5; mannose-binding lectin, metallothionein 1 and 2), 2 were highly downregulated (microsomal epoxide hydrolase 1 and cytochrome P450 1a1) . These genes were found to have high potential for immune-modulatory effects . CONCLUSIONS: The observed alterations in IEL gene expression may have an important role in the altered immune response with TPN and may relate to the increase in sepsis with TPN administration.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1028 - 37 Epub 2003 May 23.
Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome; Page R et al.; As the field of structural genomics continues to grow and new technologies are developed, novel strategies are needed to efficiently crystallize large numbers of protein targets, thus increasing output, not just throughput {Chayen & Saridakis (2002) . Acta Cryst . D58, 921-927} . One strategy, developed for the high-throughput structure determination of the Thermotoga maritima proteome, is to quickly determine which proteins have a propensity for crystal formation followed by focused SeMet-incorporated protein crystallization attempts . This experimental effort has resulted in over 320 000 individual crystallization experiments . As such, it has provided one of the most extensive systematic data sets of commonly used crystallization conditions against a wide range of proteins to date . Analysis of this data shows that many of the original screening conditions are redundant, as all of the T . maritima proteins that crystallize readily could be identified using just 23% of the original conditions . It also shows that proteins that contain selenomethionine and are more extensively purified often crystallize in distinctly different conditions from those of their native less pure counterparts . Most importantly, it shows that the two-tiered strategy employed here is extremely successful for predicting which proteins will readily crystallize, as greater than 99% of the proteins identified as having a propensity to crystallize under non-optimal native conditions did so again as selenomethionine derivatives during the focused crystallization trials . This crystallization strategy can be adopted for both large-scale genomics programs and individual protein studies with multiple constructs and has the potential to significantly accelerate future crystallographic efforts.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7678 - 83 Epub 2003 May 30.
Lateral gene transfer and the evolution of plastid-targeted proteins in the secondary plastid-containing alga Bigelowiella natans; Archibald JM et al.; Chlorarachniophytes are amoeboflagellate algae that acquired photosynthesis secondarily by engulfing a green alga and retaining its plastid (chloroplast) . An important consequence of secondary endosymbiosis in chlorarachniophytes is that most of the nuclear genes encoding plastid-targeted proteins have moved from the nucleus of the endosymbiont to the host nucleus . We have sequenced and analyzed 83 cDNAs encoding 78 plastid-targeted proteins from the model chlorarachniophyte Bigelowiella natans (formerly Chlorarachnion sp . CCMP621) . Phylogenies inferred from the majority of these genes are consistent with a chlorophyte green algal origin . However, a significant number of genes ( approximately 21%) show signs of having been acquired by lateral gene transfer from numerous other sources: streptophyte algae, red algae (or algae with red algal endosymbionts), as well as bacteria . The chlorarachniophyte plastid proteome may therefore be regarded as a mosaic derived from various organisms in addition to the ancestral chlorophyte plastid . In contrast, the homologous genes from the chlorophyte Chlamydomonas reinhardtii do not show any indications of lateral gene transfer . This difference is likely a reflection of the mixotrophic nature of Bigelowiella (i.e., it is photosynthetic and phagotrophic), whereas Chlamydomonas is strictly autotrophic . These results underscore the importance of lateral gene transfer in contributing foreign proteins to eukaryotic cells and their organelles, and also suggest that its impact can vary from lineage to lineage.

Mol Biol Evol, 2003 Jul, 20(7), 1134 - 42 Epub 2003 May 30.
Retroids in archaea: phylogeny and lateral origins; Rest JS et al.; Until recently, none of the diverse elements bearing reverse transcriptase (retroids) have been known from Archaea . However, in the recently published genomes of the acetate-utilizing archaeal methanogens, Methanosarcina acetivorans and M . mazei, several open reading frames (ORFs) are annotated as reverse transcriptase (RT) . These annotations led us to the characterization of a retron and 13 retrointrons, including three twintrons, clustered at seven loci of the M . acetivorans genome, and four retrointrons at two loci of the M . mazei genome . Based on a phylogeny of the RT ORFs, we infer four lateral gene transfers (LGT) of these retroids from Bacteria to Archaea and of retrointron mobility within the Archaea genomes . Our phylogenetic analysis also identifies several novel retrons from GenBank in the bacterial groups Firmicutes, Fusobacteria, Cyanobacteria and beta-Proteobacteria, as well as in M . acetivorans . The discovery of retrointrons in Archaea as a consequence of LGT from Bacteria suggests that they did not originate in the progenote and parallels the "mitochondrial seed" theory of the origin of spliceosomes . Extending the known phylogenetic distribution of retroids to Archaea is consistent with the view that they have played a significant role in evolution of genomes throughout the tree of life.

Microbiology, 2003 Jun, 149(Pt 6), 1593 - 603
Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene; Dziadek J et al.; To understand the role of Mycobacterium smegmatis ftsZ (ftsZ(smeg)) in the cell division process, the ftsZ gene was characterized at the genetic level . This study shows that ftsZ(smeg) is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy . Expression of ftsZ(smeg) in M . smegmatis from a constitutively active mycobacterial promoter resulted in lethality whereas that from a chemically inducible acetamidase (ami) promoter led to FtsZ accumulation, filamentation and cell lysis . To further understand the roles of ftsZ in cell division a conditionally complementing ftsZ(smeg) mutant strain was constructed in which ftsZ expression is controlled by acetamide . Growth in the presence of 0.2 % acetamide increased FtsZ levels approximately 1.4-fold, but did not decrease viability or change cell length . Withdrawal of acetamide reduced FtsZ levels, decreased viability, increased cell length and eventually lysed the cells . Finally, it is shown that ftsZ(smeg) function in M . smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZ(tb) can independently initiate the formation of Z-rings and catalyse the septation process . It is concluded that optimal levels of M . smegmatis FtsZ are required to sustain cell division and that the cell division initiation mechanisms are similar in mycobacteria.

Microbiology, 2003 Jun, 149(Pt 6), 1367 - 75
Arming the enemy: the evolution of resistance to self-proteins; Bell G et al.; A remarkable range of novel antibiotics is attracting increasing interest as a major new weapon in the campaign against bacterial infection . They are based on the toxic peptides that provide the innate immune system of animals, and it is claimed that bacteria will be unable to evolve resistance to them because they attack the 'Achilles' heel' of bacterial membrane structure . Both experimental evidence and theoretical arguments suggest that this claim is doubtful . If so, the introduction of these substances into general use may provoke the evolution of resistance to our own defence proteins and thus compromise our natural defences against infection.

Arch Immunol Ther Exp (Warsz), 2003, 51(2), 131 - 6
Serological differentiation of Helicobacter pylori CagA(+) and CagA(-) infections; Chmiela M et al.; Many Helicobacterpylori strains causing gastroduodenal diseases have a cagA gene encoding CagA protein, a virulence factor of these bacteria . Anti-CagA antibodies produced by the majority of people infected with CagA(+) strains can indicate such an infection . In this study, the efficacy of three immunoenzymatic tests for detecting CagA(+) and CagA(-) infections were compared: immunoblot (Milenia ID Blot H . pylori IgG; MB) and ELISA conducted either with a recombinant immunodominant fragment of CagA (rCagA) or the full-length CagA molecule (flCagA) . The 13C-urea breath test (13C-UBT) was used for establishing H . pylori status . The serum samples from 157 individuals were used for serodiagnosis . H . pylori CagA(+) infection was detected in H . pylori-infected individuals with similar frequencies by MB (64%) and flCagA-ELISA (60%) and a little less frequently by rCagA-ELISA (53%) . There was a high coincidence between the negative results of these three tests for H . pylori-uninfected individuals with no anti-CagA IgG in the serum (96-100%) . The results show that rCagA-ELISA and, especially, flCagA-ELISA are easy, inexpensive and useful noninvasive assays for the discrimination of CagA(+) and CagA(-) H . pylori infections in subjects examined by urea breath test.

Nat Rev Immunol, 2003 Jun, 3(6), 445 - 53
TREMs in the immune system and beyond; Colonna M; Triggering receptors expressed by myeloid cells (TREMs) belong to a rapidly expanding family of receptors that include activating and inhibitory isoforms encoded by a gene cluster linked to the MHC . TREM1 and TREM2 activate myeloid cells by signalling through the adaptor protein DAP12 . TREM1 triggers phagocyte secretion of pro-inflammatory chemokines and cytokines, amplifying the inflammation that is induced by bacteria and fungi . TREM2 activates monocyte-derived dendritic cells and regulates osteoclast development . Remarkably, TREM2 deficiency leads to a severe disease that is characterized by bone cysts and demyelination of the central nervous system, which results in dementia, implying that the function of TREM2 extends beyond the immune system.

J Bacteriol, 2003 Jun, 185(12), 3678 - 82
Xenorhabdus nematophila requires an intact iscRSUA-hscBA-fdx operon to colonize Steinernema carpocapsae nematodes; Martens EC et al.; An insertion between iscA and hscB of the Xenorhabdus nematophila iscRSUA-hscBA-fdx locus, predicted to encode Fe-S assembly machinery, prevented colonization of Steinernema carpocapsae nematodes . The insertion disrupted cotranscription of iscA and hscB, but did not reduce hscBA expression, suggesting that X . nematophila requires coordinated expression of the isc-hsc-fdx locus for colonization.

J Bacteriol, 2003 Jun, 185(12), 3668 - 71
Involvement of cyclic AMP (cAMP) and cAMP receptor protein in anaerobic respiration of Shewanella oneidensis; Saffarini DA et al.; Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides . Two S . oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed . Both mutants were deficient in Fe(III) and Mn(IV) reduction . They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO . Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor . This growth deficiency may be due to the observed loss of cytochromes c in the mutants . In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP) . Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S . oneidensis . These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.

Int J Environ Health Res, 2003 Jun, 13 Suppl 1, S67 - 72
Housing improvement projects in Indonesia: responding to local demand; Josodipoero RI; For more than three decades, environmental health programmes in Indonesia have emphasized prevention and treatment of the high incidence of disease among villagers . One of the main causes of disease is the unhygienic conditions of typical rural houses - two-room constructions with dirt floors and walls of lightly fired bricks or woven bamboo skins . While most houses have few or no windows, the occupants frequently cook, eat, sleep and even keep animals in a single room . The main objective of the housing improvement programme was to improve air circulation and introduce more sunlight to kill bacteria, avoid dampness and eliminate smoke from cooking . The programme encourages villagers to construct a permanent floor, enlarge existing windows or insert new windows for good ventilation . This presentation will share the 'success stories' of housing improvement projects in Indonesia that adopted demand-responsive approaches instead of the conventional 'supply approach' . Through exercises like Wealth Classification and Social Mapping, a demand-responsive approach lets the community decide who is eligible for assistance, resulting in higher participation and accurate information on community demand and on materials needed . In addition to the successes, the failures will be discussed at field level . This presentation will discuss the lessons learned from: the World Bank-funded Kalisemut Case Study; government's Family Welfare Movement; Plan International's project in Yogyakarta, and AusAID-funded Sustainable Development through Community Participation Project in Lombok.

BMC Bioinformatics . 2003 May 29;4(1):20.
Information extraction from full text scientific articles: where are the keywords?
Shah PK, Perez-Iratxeta C, Bork P, Andrade MA.
BACKGROUND: To date, many of the methods for information extraction of biological information from scientific articles are restricted to the abstract of the article . However, full text articles in electronic version, which offer larger sources of data, are currently available . Several questions arise as to whether the effort of scanning full text articles is worthy, or whether the information that can be extracted from the different sections of an article can be relevant . RESULTS: In this work we addressed those questions showing that the keyword content of the different sections of a standard scientific article (abstract, introduction, methods, results, and discussion) is very heterogeneous . CONCLUSIONS: Although the abstract contains the best ratio of keywords per total of words, other sections of the article may be a better source of biologically relevant data.

J Endod, 2003 May, 29(5), 321 - 3
Cytomegalovirus and Epstein-Barr virus active infection in periapical lesions of teeth with intact crowns; Sabeti M et al.; Herpesviruses seem to play an important role in the pathogenesis of aggressive periodontitis and may also contribute to periapical pathosis . This study determined the presence of human cytomegalovirus, Epstein-Barr virus, and herpes simplex virus productive infection in five symptomatic periapical lesions of teeth having intact crowns and calcified necrotic pulps . Periapical samples were collected in conjunction with periapical surgery and kept frozen until virological examination . Reverse transcription-polymerase chain reaction was used in herpesviral identification . RNA was isolated from periapical tissue by a guanidinium isothiocyanate-acid phenol procedure . cDNAs were generated from highly conserved regions of the test viruses using a preamplification kit . Sensitivity and validity of the PCR-primers were determined according to established methods . Amplification products were identified using gel electrophoresis . Human cytomegalovirus and Epstein-Barr virus dual transcription was detected in all five periapical lesions studied . Herpes simplex virus transcript was not identified in any lesion . The present data suggest that human cytomegalovirus or Epstein-Barr virus activation participate in the pathogenesis of symptomatic periapical lesions . We hypothesize that periapical active herpesvirus infection impairs local defenses, thereby inducing overgrowth of endodontopathic bacteria and the clinical flare-up of inflammation.

Zhongguo Zhong Yao Za Zhi, 2002 Jun, 27(6), 401 - 3
{Chemical and pharmacological advances of the study on zushima}; Li SH et al.; OBJECTIVE: To review the progress in the research of the active ingredients of Zushima and their pharmacological activities . METHOD: Base on the articles of the chemical constituents and pharmacological activities of Zushima . RESULT: Traditional Chinese drug, Zushima contains coumarins, diteropenoids, lignans, flavonoids, anthraquinones and sterols . Pharmacological investigation concludes that it has actions of painkilling, antiinflammation, inhibiting bacteria, antithrombus, antitumer and antifertility . CONCLUSION: Zushima has extensive actions in pharmacology . And plant resources are very rich . It is a meaning job to study the chemical ingredients and pharmacological activities of Zushima further.

Int J Surg Investig, 1999, 1(4), 301 - 6
Helicobacter pylori products upregulate neutrophil superoxide anion production; Tennenberg SD et al.; Helicobacter pylori-neutrophil interactions may play a pathogenic role in H . pylori-induced gastritis and peptic ulcer disease . To understand these interactions, we explored the effects of H . pylori-derived products on neutrophil chemotaxis and superoxide anion production . H . pylori bacteria were cultured and supernatants fractionated . Neutrophil chemotactic activity was confirmed in the crude supernatants and in one fractionated peak corresponding to a previously described neutrophil chemotactic factor . H . pylori-derived crude supernatant, sonicate and all chromatography-derived peaks failed to directly stimulate neutrophil superoxide anion production . However, after pretreatment with sonicate, neutrophils demonstrated increased superoxide anion production (priming) following subsequent exposure to the secretagogue fmet-leu-phe . These results suggest that H . pylori products may attract neutrophils to the gastric mucosa without initially stimulating superoxide anion production or tissue injury . Oxygen radical-mediated gastric mucosal injury may subsequently result when these primed neutrophils undergo additional stimulation by as yet unidentified factors.

Zhongguo Zhong Yao Za Zhi, 2002 Feb, 27(2), 139 - 43
{Effects of the zhikuofang on the inflammation and cytostatics of the airway model of bronchiectasis}; Wan YG et al.; OBJECTIVE: To explore the effects of Zhikuofang, a TCM prescription, and Ofloxacin on the inflammation and cytostatics of the airway model of bronchiectasis . METHOD: The airway model of bronchiectasis (AMB) was set up and infused with Ps . Aeruginosa . A comparison between the effects of Zhikuofang and Of loxacin on the AMB was made . RESULT: Zhikuofang is better than Ofloxacin in following aspects: lowering the density of inflammation cells in blood, decreasing the volume of tracheal secretion and inhibiting the cytostatics (IL-8 and TNF-alpha) of the trachea tissue, but Ofloxacin is more effective in diminishing the amount of bacteria in trachea flushing liquor . There was no marked difference between them in their histopathy effects on the trachea . CONCLUSION: Zhikuofang probably plays antiphlogistic and bacteriostatic effects by inhibiting the IL-8 and TNF-alpha, resisting secretion, decreasing the inflammation cells and resisting inflammation of trachea.

J Mol Model (Online), 2003 Aug, 9(4), 217 - 24 Epub 2003 May 27.
Construction of a toroidal model for the magainin pore; Murzyn K et al.; Magainins are natural peptides that selectively kill bacteria at concentrations that are harmless to animal cells . Due to a positive charge and distinct hydrophobic moment, magainins in the alpha-helical conformation interact favorably with bacterial membrane lipids . These interactions lead to the formation of large openings in the membrane and to the cell's death . The openings (toroidal pores) are supramolecular structures consisting of lipid and peptide molecules . A computer model of the pore in a bacterial membrane was constructed (see Figure) for the study of the molecular basis for magainin selectivity and specificity . Details of the construction and the preliminary equilibration of the pore model are given in this paper.FIGURE Stages of PORE construction.A A side view andB the top view of E(6) cluster (see text for details) . The coloring scheme of lipid molecules is described in the caption to Fig . 5.C The top view of the magainin pore built of five M2a molecules (in purple and gold), five EG(4)E clusters, and five E(6) clusters.D The top view of the lamellar part of PORE . The size of the hole in the bilayer center matches the size of the supramolecular complex shown inC.E The pore (C) is now placed in the central part of the bilayer (D)

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7307 - 12 Epub 2003 May 28.
Decorin-binding proteins A and B confer distinct mammalian cell type-specific attachment by Borrelia burgdorferi, the Lyme disease spirochete; Fischer JR et al.; Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types . Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B . burgdorferi surface proteins . We previously showed that B . burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind . The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen . In the current study, we show that the expression of decorin-binding protein (Dbp) A and/or DbpB, two B . burgdorferi surface proteins that bind GAGs, is sufficient to convert a high-passage nonadherent B . burgdorferi strain into one that efficiently binds 293 epithelial cells . Epithelial cell attachment was mediated by dermatan sulfate, and, consistent with this GAG-binding specificity, these recombinant strains did not bind EA-Hy926 endothelial cells . The GAG-binding properties of bacteria expressing DbpB or DbpA were distinguishable, and DbpB but not DbpA promoted spirochetal attachment to C6 glial cells . Thus, DbpA and DbpB may each play central but distinct roles in cell type-specific binding by Lyme disease spirochetes . This study illustrates that transformation of high-passage B . burgdorferi strains may provide a relatively simple genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.

J Leukoc Biol, 2003 Jun, 73(6), 702 - 12
Nondisposable materials, chronic inflammation, and adjuvant action; Hamilton JA; Why inflammatory responses become chronic and how adjuvants work remain unanswered . Macrophage-lineage cells are key components of chronic inflammatory reactions and in the actions of immunologic adjuvants . One explanation for the increased numbers of macrophages long term at sites of chronic inflammation could be enhanced cell survival or even local proliferation . The evidence supporting a unifying hypothesis for one way in which this macrophage survival and proliferation may be promoted is presented . Many materials, often particulate, of which macrophages have difficulty disposing, can promote monocyte/macrophage survival and even proliferation . Materials active in this regard and which can initiate chronic inflammatory reactions include oxidized low-density lipoprotein, inflammatory microcrystals (calcium phosphate, monosodium urate, talc, calcium pyrophosphate), amyloidogenic peptides (amyloid beta and prion protein), and joint implant biomaterials . Additional, similar materials, which have been shown to have adjuvant activity (alum, oil-in-water emulsions, heat-killed bacteria, CpG oligonucleotides, methylated bovine serum albumin, silica), induce similar responses . Cell proliferation can be striking, following uptake of some of the materials, when macrophage-colony stimulating factor is included at low concentrations, which normally promote mainly survival . It is proposed that if such responses were occurring in vivo, there would be a shift in the normal balance between cell survival and cell death, which maintains steady-state, macrophage-lineage numbers in tissues . Thus, there would be more cells in an inflammatory lesion or at a site of adjuvant action with the potential, following activation and/or differentiation, to perpetuate inflammatory or antigen-specific, immune responses, respectively.

EMBO J, 2003 Jun 2, 22(11), 2561 - 70
Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein; Kerr ID et al.; Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria . Single-stranded DNA-binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways . However, whilst euryarchaea have a eukaryotic-type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C-terminal tail that is implicated in protein-protein interactions . We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 A . The structure shows a striking and unexpected similarity to the DNA-binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes . The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.

Am J Physiol Cell Physiol, 2003 Oct, 285(4), C968 - 76 Epub 2003 May 28.
Plasma membrane delivery of the gastric H,K-ATPase: the role of beta-subunit glycosylation; Vagin O et al.; The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive . To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase beta-subunit (YFP-beta) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase alpha-subunit (CFP-alpha) were expressed in HEK-293 cells . Then plasma membrane delivery of wild-type CFP-alpha, wild-type YFP-beta, and YFP-beta mutants lacking one or two of the seven beta-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation . Expression of the wild-type YFP-beta resulted in the plasma membrane localization of the protein, whereas the expressed CFP-alpha was retained intracellularly . When coexpressed, both CFP-alpha and YFP-beta were delivered to the plasma membrane . Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-beta prevented plasma membrane delivery of the protein . Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane . A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein . Therefore, six of the seven glycosylation sites in the beta-subunit are essential for the plasma membrane delivery of the beta-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the beta-subunits from different species, is not critical for plasma delivery of the protein.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 703 - 5
Homologous recombination in Archaea: new Holliday junction helicases; Bolt EL et al.; Homologous recombination (HR) maintains genome stability by promoting high fidelity DNA repair . Several recent reports have established that the primary function of HR enzymes is to underpin DNA replication, resetting forks that are blocked or collapsed at sites of DNA damage remote from replication origins . These functions are crucial to ensuring that genomes are transmitted successfully into subsequent generations of cells . Enzymes of HR have been unearthed in all three domains of life: bacteria, Archaea and eukarya . Helicases that specifically unwind branched DNA molecules are pivotal in linking HR and DNA replication in bacteria . However, knowledge of helicases with these functions in eukaryotes is vague and is wholly absent in Archaea . We are using the archaeal species Methanothermobacter thermautotrophicus to identify new DNA helicases of homologous recombination.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 694 - 8
Repair of UV damage in Halobacterium salinarum; McCready S et al.; Halobacterium is one of the few known Archaea that tolerates high levels of sunlight in its natural environment . Photoreactivation is probably its most important strategy for surviving UV irradiation and we have shown that both of the major UV photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts, can be very efficiently repaired by photoreactivation in this organism . There are two putative photolyase gene homologues in the published genome sequence of Halobacterium sp . NRC-1 . We have made a mutant deleted in one of these, phr2, and confirmed that this gene codes for a CPD photolyase . (6-4) photoproducts are still photoreactivated in the mutant so we are currently establishing whether the other homologue, phr1, codes for a (6-4) photolyase . We have also demonstrated an excision repair capacity that operates in the absence of visible light but the nature of this pathway is not yet known . There is probably a bacteria-type excision-repair mechanism, since homologues of uvrA, uvrB, uvrC and uvrD have been identified in the Halobacterium genome . However, there are also homologues of eukaryotic nucleotide-excision-repair genes ( Saccharomyces cerevisiae RAD3, RAD25 and RAD2 ) so there may be multiple repair mechanisms for UV damage in Halobacterium.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 690 - 3
Archaeal DNA repair: paradigms and puzzles; White MF; It is now generally accepted that the Archaea share many similarities in their information-processing pathways with eukarya . Archaeal and eukaryal DNA replication and transcriptional machineries show particularly striking similarities, and the archaeal processes have been used extensively as simpler models of the much more complex eukaryal ones . Archaeal DNA-repair pathways are not yet well characterized, and their relationship with repair pathways in bacteria and eukarya are still open to question . There are also strong distinctions between the major subdivisions crenarchaea and euryarchaea within the archaeal domain . This review highlights some of these similarities and differences using specific examples arising from our studies of the double-stranded and single-stranded DNA-binding proteins and the repair endonuclease XPF in the crenarchaeote Sulfolobus solfataricus.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 548 - 52
Experiences with the shikimate-pathway enzymes as targets for rational drug design; Coggins JR et al.; The background and current context of work on the shikimate-pathway enzymes as potential targets for anti-bacterial, anti-fungal and anti-parasitic drugs is reviewed . Recent work on the third enzyme of the pathway, dehydroquinase, which occurs in two structurally and mechanistically distinct forms, is used to illustrate the present state of studies into rational drug design.

J Med Chem, 2003 Jun 5, 46(12), 2542 - 7
Elicitation, a new window into plant chemodiversity and phytochemical drug discovery; Poulev A et al.; Plant extracts collected from the wild are important sources for drug discovery . However, these extracts suffer from a lack of reproducible bioactivity and chemical composition caused by the highly inducible, variable, and transitory nature of plant secondary metabolism . Here, we demonstrate that exposing roots of hydroponically grown plants to chemical elicitors selectively and reproducibly induced the production of bioactive compounds, dramatically increased the hit rate, and more than doubled the number of plant species showing in vitro activity against bacteria, fungi, or cancer . Elicitation performed under controlled conditions dramatically improves reliability and efficiency of plant extracts in drug discovery while preserving wild species and their habitats.

Clin Tech Small Anim Pract, 2003 Feb, 18(1), 39 - 44
Cytology and fluid analysis of the acute abdomen; Connally HE; In patients with acute abdominal pain, abdominal paracentesis and diagnostic peritoneal lavage often yield fluid samples for cytologic and biochemical evaluation . Cytology of the effusion from a patient with acute abdominal disease can be a crucial tool for the rapid diagnosis necessary for initiation of timely and appropriate therapy . Appropriate sample collection, handling, and preparation are essential to obtain an accurate diagnosis . Analysis of the fluid sample should include gross examination of the effusion, measurement of total nucleated cell count, packed red blood cell volume, and protein concentration, as well as examination for the presence of other cells, bacteria, food particles, or plant material . Biochemical evaluation should proceed based on the clinician's index of suspicion for a particular disease process . Abdominal effusions are generally classified as transudate, modified transudate, or exudate, depending on the total nucleated cell count and protein concentration . Cytology of all fluids collected should be performed systematically, utilizing progressively higher magnifications with a microscope . Specific diseases with associated abdominal effusions include septic peritonitis, nonseptic peritonitis, hemoabdomen, uroabdomen, pancreatitis, bile peritonitis, chylous effusion, and neoplasia . A complete description of sample preparation and evaluation is reviewed.

Eye Contact Lens, 2003 Jan, 29(1 Suppl), S10 - 3; discussion S26-9, S192-4
Innate defense of the ocular surface; McNamara NA; PURPOSE: The mucosal surface of the eye interfaces with the environment and can detect the presence of pathogens and irritants . The mucosal cells respond by up-regulating genes whose products either directly attack the contaminant or recruit leukocytes through the release of cytokines . Mucin, an epithelial glycoprotein, traps contaminants and aids in their removal through normal tear clearance mechanisms . METHODS AND RESULTS: Using Northern analysis and reverse transcription-polymerase chain reaction, we have found that MUC1, a membrane-bound ocular surface mucin, is up-regulated following exposure to the products of both bacteria and activated lymphocytes . Consistent with this, MUC1 gene expression is also up-regulated in corneal epithelial cells exposed to precorneal tear film collected from two rigid contact lens wearers . CONCLUSION: The changes in MUC1 gene expression during exposure to potential ocular irritants suggest that analysis of the molecular mechanisms mediating these changes may reveal control points amenable to drug therapy.

Nihon Kokyuki Gakkai Zasshi, 2003 Mar, 41(3), 181 - 5
{A case of pulmonary actinomycosis with recurrent hemoptysis}; Nishimoto T et al.; A 68-year-old man was admitted to our hospital because of hemoptysis in September 1999 . Chest CT scans showed a nodular shadow with infiltration in the right S 7 . Bronchial arteriography showed vascularization in the right S 7, and bronchial artery embolization was performed . However, in April and October 2000 hemoptysis recurred, and bronchial arteriography showed recurrence of vascularization in the same area, so embolization was performed again . Then, the patient was admitted in March 2001 because of recurrent hemoptysis . CT scans showed growth of the nodular shadow . Right lower lobectomy was performed, and the microscopic findings in the tissue from the resected lobe showed branching filamentous bacteria, and pulmonary actinomycosis was diagnosed . We concluded that pulmonary actinomycosis should be considered in the differential diagnosis of nodular shadows with recurrent hemoptysis.

Curr Opin Mol Ther, 2003 Apr, 5(2), 123 - 32
Peptide-assisted delivery of steric-blocking antisense oligomers; Moulton HM et al.; Cationic transport peptides conjugated to steric blocking antisense oligomers (oligos) increase oligo uptake in eukaryotic cell lines, bacteria and mice . Recent reports of arginine-rich transport peptide conjugates strongly suggest that the mechanism of uptake is primarily endocytotic and that previous assay techniques produced confounding artifacts that led to the old non-endocytotic, membrane-penetrating peptide model . The artifacts result from fixing cells for fluorescent microscopy and from using non-trypsinized cells for flow cytometry . Fixing cells redistributes the peptide or peptide-oligo conjugates associated with the outside of cell membranes and trapped in endosomes, giving apparent diffuse cytosolic and nuclear fluorescence . Cationic peptides bound to the outer surface of cells, if not removed, skew fluorescence data obtained by flow cytometry, leading to the earlier conclusions . Upregulation assays now provide a tool for comparing the efficacy of conjugates, measuring oligo uptake by quantitating antisense activity of conjugates . These assays, developed in cell culture and mouse models, are faster and have higher signal-to-noise ratios than downregulation assays . Thus, a convenient and effective method now exists to screen transport peptides.

Pediatr Pulmonol, 2003 Jul, 36(1), 58 - 62
Sputum induction as a diagnostic tool for community-acquired pneumonia in infants and young children from a high HIV prevalence area; Zar HJ et al.; Sputum induction is a standard diagnostic procedure to identify pathogens in lower respiratory tract secretions in adults with pneumonia, but has rarely been studied or used in infants and young children . Our aim was to determine the usefulness of induced sputum (IS) as a diagnostic method for infants and children hospitalized with community-acquired pneumonia (CAP) in a high HIV prevalence area . Children hospitalized for CAP were prospectively enrolled over a year . IS was obtained by nebulization with hypertonic (5%) saline, physiotherapy, and suctioning . Sputum was submitted for bacterial and mycobacterial culture and P . carinii detection . Gastric lavages (GLs) were done for M . tuberculosis culture; a nasopharyngeal aspirate (NPA) was obtained for bacterial culture and P . carinii detection . IS was obtained in 210 children (median age, 7 (25th to 75th percentile, 3-18) months); 138 (66%) were HIV-infected; 148 (70%) were receiving supplemental oxygen . Bacteria were isolated from 101 (50%) IS and 141 (70%) NPA paired specimens (P < 0.001) . A significantly higher rate of S . aureus, H . influenzae, M . catarrhalis, and S . pneumoniae was found in NPAs compared to IS; this pattern was particularly evident in HIV-infected children . M . tuberculosis was cultured from sputum in 19 patients (9%); GLs performed in 142 children were positive in only 9 (6%) . The difference (95% confidence interval) between yields for M . tuberculosis from culture of IS compared to GL was 4.3% (95% CI, 0-5.6%; P = 0.08) . P . carinii was identified from IS in 12 (5.7%) children; all corresponding NPAs were negative . Seven (3%) children could not tolerate sputum induction . Side effects included increased coughing in 4%, epistaxis in 3%, and wheezing responsive to bronchodilators in 1% . In conclusion, induced sputum is a useful and safe diagnostic procedure in infants and children with CAP from a high HIV prevalence area .

Occup Environ Med, 2003 Jun, 60(6), 444 - 50
Upper airway inflammation in waste handlers exposed to bioaerosols; Heldal KK et al.; AIMS: To examine work associated upper airway inflammation in 31 waste handlers, and to correlate these findings with personally monitored exposure to different bioaerosol components . METHODS: Cell differentials, interleukin 8 (IL-8), myeloperoxidase (MPO), and eosinophilic cationic protein (ECP) were examined in NAL (nasal lavage), and swelling of the nasal mucosa was determined by acoustic rhinometry before work start on Monday and the following Thursday . Bioaerosol exposure was determined by personal full shift exposure measurements on Monday, Tuesday, and Wednesday and analysed for total bacteria, fungal spores, endotoxin, and beta(1-->3)-glucans . RESULTS: The increased percentage of neutrophils from Monday (28%) to Thursday (46%) correlated with increases in ECP (r(S) = 0.71, p < 0.001) and MPO (r(S) = 0.38, p < 0.05), and showed a close to significant correlation with nasal swelling (r(S) = -0.55, p = 0.07) . The Thursday levels of neutrophils, MPO, and IL-8 were associated with the exposure to fungal spores (range 0-2.0 x 10(6)/m(3)) and endotoxin (range 4-183 EU/m(3)) measured the day before, and the median exposure to beta(1-->3)-glucans (range 3-217 ng/m(3)), respectively (r(S) = 0.47-0.54, p < 0.01) . Swelling of the nasal mucosa was associated with the fungal spore and beta(1-->3)-glucan exposure (r(S) = 0.58-0.59, p < 0.05) . CONCLUSION: These results are based on a relatively small population, and conclusions must be drawn with care . The results suggested that a moderate exposure to fungal spores, endotoxins, and beta(1-->3)-glucans during waste handling induced upper airway inflammation dominated by neutrophil infiltration and swelling of the nasal mucosa.

Nucleic Acids Res . 2003 Jun 1;31(11):e59.
A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein; Kramer RA et al.; Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules . A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets . This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency . Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII . Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection . Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage . The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target . We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.

J Insect Physiol, 2003 May, 49(5), 419 - 32
Origin and evolution of polydnaviruses by symbiogenesis of insect DNA viruses in endoparasitic wasps; Federici BA et al.; During oviposition, many endoparasitic wasps inject virus-like particles into their insect hosts that enable these parasitoids to evade or directly suppress their hosts' immune system, especially encapsulation by hemocytes . These particles are defined as virions that belong to viruses of the two genera that comprise the family Polydnaviridae, bracoviruses (genus Bracovirus) transmitted by braconid wasps, and ichnoviruses (genus Ichnovirus) transmitted by ichneumonid wasps . Structurally, bracovirus virions resemble nudivirus and baculovirus virions (family Baculoviridae), and ichnovirus virions resemble those of ascoviruses (family Ascoviridae) . Whereas nudiviruses, baculoviruses and ascoviruses replicate their DNA and produce progeny virions, polydnavirus DNA is integrated into and replicated from the wasp genome, which also directs virion synthesis . The structural similarity of polydnavirus virions to those of viruses that attack the wasps' lepidopteran hosts, along with polydnavirus transmission and replication biology, suggest that these viruses evolved from insect DNA viruses by symbiogenesis, the same process by which mitochondia and chloroplasts evolved from bacteria . Molecular evidence supporting this hypothesis comes from similarities among structural proteins of ascoviruses and the Campoletis sonorensis ichnovirus . Implications of this hypothesis are that polydnaviruses evolved from viruses, but are no longer viruses, and that DNA packaged into polydnavirus virions is not viral genomic DNA per se, but rather wasp genomic DNA consisting primarily of wasp genes and non-coding DNA . Thus, we suggest that a better understanding of polydnaviruses would result by viewing these not as viruses, but rather as a wasp organelle system that evolved to shuttle wasp genes and proteins into hosts to evade and suppress their immune response.

Biologicals, 2003 Jun, 31(2), 107 - 12
BVDV and innate immunity; Peterhans E et al.; Infection with bovine viral diarrhea virus (BVDV) is prevalent in the cattle population worldwide . The virus exists in two biotypes, cytopathic and non-cytopathic, depending on the effect of the viruses on cultured cells . BVDV may cause transient and persistent infections which differ fundamentally in the host's antiviral immune response . Transient infection may be due to both cytopathic and non-cytopathic biotypes of BVDV and leads to a specific immune response . In contrast, only non-cytopathic BVD viruses can establish persistent infection as a result of infection of the embryo early in its development . Persistent infection is characterized by immunotolerance specific for the infecting viral strain . In this paper we discuss the role of innate immune responses in the two types of infection . In general, both transient and persistent infections are associated with an increased frequency of secondary infections . Associated with the increased risk of such infections are, among others, impaired bacteria killing and decreased chemotaxis . Interestingly, non-cytopathic BVDV fails to induce interferon type I in cultured bovine macrophages whereas cytopathic biotypes readily trigger this response . Cells infected with non-cytopathic BVDV are also resistant to induction of interferon by double stranded RNA, a potent interferon inducer signalling the presence of viral replication in the cell . Thus, non-cytopathic BVDV may dispose of a mechanism suppressing a key element of the antiviral defence of the innate immune system . Since interferon is also important in the activation of the adaptive immune response, suppression of this signal may be essential for the establishment of persistent infection and immunotolerance.

J Insect Physiol, 1999 Jul, 45(7), 677 - 685
Insect immunity-effects of factors produced by a nematobacterial complex on immunocompetent cells(1); Ribeiro C et al.; During in vitro incubations, the nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophilus produces different factors having toxic activities in vitro towards haemocytes, the insect cells responsible for cellular immune defense reactions . Among others, two effects were evident on haemocyte monolayers; one of them was a cytotoxic activity while the other was an unsticking effect . The factors responsible for cytotoxic activity and unsticking effect, were separated from each other by a single chromatography on anion exchange column . These two effects on haemocytes were lost after heat treatment at 57 degrees C for 1 h and 45 degrees C for 30 min, respectively . Both factors were recovered after dialysis in a 10(4) Da cut off membrane . The cytotoxic activity was susceptible to proteases . Cytotoxic and unsticking factors did not show any lipase or lecithinase activity but the unsticking factor had protease activity . Lipopolysaccharides, purified from the bacteria harvested after incubation of the complex, did not have cytotoxic or unsticking effect on the insect cells in vitro.

J Insect Physiol, 2000 Apr, 46(4), 605 - 610
Exuviae eating: a nitrogen meal?
Mira A.
Many insects eat their cast cuticle (exuviae) after moulting . The functional significance of this behaviour has not been addressed experimentally . I tested the hypothesis that exuviae eating constitutes a meal, so the animal recycles its nitrogen content . Nitrogenous compounds (protein and chitin) are major components of the cuticle in Periplaneta americana, accounting for as much as 87% of the total weight . It was found that insects almost invariably ate their exuviae during their larval life . The frequency of the behaviour decreased in newly emerged adults and varied between the sexes, males eating their exuviae less frequently than females . This may be due to the extra nitrogen endowment which females need for reproduction . Aposymbiotic animals, which lack the supply of essential amino acids from endosymbiotic bacteria, always ate their exuviae regardless of sex . When animals were reared on different diets throughout their larval life protein level in the diet correlated with exuviae eating . Animals reared on a low protein diet showed the highest levels of exuviae eating; animals reared on a high protein diet showed the highest levels of exuviae rejection . Analysis of the frass produced after exuviae meals showed that over 58% of the nitrogen present in the exuviae was recycled . This demonstrated that cockroaches digested nitrogenous compounds contained in the cuticle . The possibility that the exuviae meal has other functions is discussed, although the evidence supports a nutritional role.

J Insect Physiol, 2002 May, 48(5), 507 - 516
Metabolic and symbiotic interactions in amino acid pools of the pea aphid, Acyrthosiphon pisum, parasitized by the braconid Aphidius ervi; Rahbe Y et al.; Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) . This parasitoid strongly redirects host reproduction and metabolism to favour nutrition and development of its juvenile stages . Parasite-regulated biosynthesis and mobilization of nitrogen metabolites determine a significant increase of host nutritional suitability . The aim of the present study was mainly to investigate the temporal changes of A . pisum amino acid pools, as affected by A . ervi parasitism, and to assess the role of the aphid bacterial endosymbiont Buchnera in determining the observed changes . In parasitized aphids, we observed a very significant increase in total free amino acids, compared with synchronous non-parasitized controls, starting from day 4 after parasitization (+51%) . This trend culminated with more than doubling the control value (+152%) on day 6 after parasitization . However, a significant "parasitism" effect was observed only for 10 of the 28 amino acids detected . Tyrosine accumulation was the most prominent parasitoid-induced alteration, with a fourfold increase over control levels registered on day 6 . In parasitized hosts, the amino acid biosynthetic capacity of Buchnera was unaltered, or even enhanced for the phenolic pool, and contributed greatly to the definition and maintainance of host free amino acid pools . The hypertyrosinemic syndrome was not dependent on food supply of the aromatic nucleus but was induced by parasitism, which likely enhanced the aromatic shuttle mediating phenylalanine transfer from bacteria to the host tissues, where tyrosine conversion occurs . This process is likely associated with a selective disruption of the host's functions requiring tyrosine, leading to the remarkable accumulation of this amino acid . The possible mechanisms determining these parasitism-induced host alterations, and their nutritional significance for the developing parasitoid larva, are discussed.

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 303 - 15 Epub 2003 May 27.
Biotechnological applications of green fluorescent protein; March JC et al.; Since its first use as a reporter gene in 1994, green fluorescent protein (GFP) has served as the researcher's agent: slipping, virtually undetected, into unseen spaces, reporting back valuable information, and securing the delivery of precious cargo through hostile domains . GFP's strength lies in its small size, formidable stability, and relative ease of use . It requires only oxygen and an energy source to do its work, which can be supplied at low cost and high precision, respectively . With such a low threshold for use, GFP is often the first line of inquiry into an unknown space . Here is provided a brief compendium of GFP's contributions to biotechnology . They are linked by a need for a level of information that was previously inaccessible, both spatially and temporally . Protein fusions, transcriptional reporters, whole-organism visualization, and various other screening applications are reviewed with respect to biotechnological applications . Germane molecular improvements to GFP are also discussed.

Spine, 2003 May 15, 28(10), 1036 - 42
Sagittal alignment after anterior debridement and fusion with or without additional posterior instrumentation in the treatment of pyogenic and tuberculous spondylodiscitis; Klockner C et al.; STUDY DESIGN: A retrospective clinical study investigated patients undergoing surgery for destructive pyogenic and tuberculous spondylodiscitis . OBJECTIVE: To compare anterior debridement and bone grafting with a combined anterior and posterior procedure in terms of the physiologic alignment of the segmental sagittal spinal profile . SUMMARY OF BACKGROUND DATA: There is considerable agreement in the literature on the indications for surgical treatment of destructive spondylodiscitis . An anterior approach usually is recommended for debridement and bone grafting . Additional posterior instrumentation is applied to reduce kyphotic deformities and to prevent a correction loss . No comparison has been made so far in the literature between repositioning results obtained after surgery for destructive spondylodiscitis and physiologic segmental sagittal angles . METHODS: The surgical results of 49 patients treated by anterior debridement and bone grafting were compared with those of 22 patients who received additional posterior instrumentation . A comparison between the segmental kyphotic angles obtained and the standard values reported in the literature enabled an assessment of the segmental spinal alignment in the sagittal plane . Data were obtained from medical record review, imaging procedures, and patient follow-up examinations . RESULTS: All the subgroups submitted to a combined procedure had a greater preoperative segmental kyphosis angle than those undergoing anterior fusion alone . In marked segmental kyphotic false positioning, good postoperative repositioning was achieved by the combined procedure, and an increase in segmental kyphosis was permanently prevented . CONCLUSIONS: In single-level spondylodiscitis with no major substance loss, anterior debridement and bone grafting alone seem to be adequate, especially in the lumbar spine . Additional posterior instrumentation is indicated in multiple-level spondylodiscitis, extensive kyphotic deformity, or both.

Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 1067 - 72
Mutational and sequence analysis of transmembrane segment 6 orientation in TetA proteins; Lewis GS et al.; The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established . However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined . In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity . The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.

Biochem Biophys Res Commun, 2003 Jun 13, 305(4), 970 - 3
DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues; Wang PL et al.; In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected . Gingival fibroblasts are the major constituents of gingival tissue . We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues {Biochem . Biophys . Res . Commun . 273 (2000) 1161} . To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis . We also studied the expression levels of these proteins by flow cytometric analysis (FACS) . The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis . FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group . Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease . Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.

Int J Cardiol, 2003 Jun, 89(2-3), 273 - 9
Prevalence of Chlamydia pneumoniae in the atherosclerotic plaque of patients with unstable angina and its relation with serology; Zamorano J et al.; BACKGROUND: Chlamydia pneumoniae has been associated with coronary artery disease by both seroepidemiological studies, and by direct detection of the micro-organism in atherosclerotic lesions . This bacteria could play a potential role in the development of acute coronary events . We examined coronary arteries from patients with unstable angina in order to verify an endovascular presence of C . pneumoniae, and to determine if there is any relationship between serology of acute infection by this pathogen and its presence inside the atherosclerotic plaque of these patients . METHODS: We analysed a total of 76 atherosclerotic plaques obtained from 45 patients who underwent coronary artery bypass surgery . In all patients unstable angina was present within the prior 3 weeks . The presence of C . pneumoniae in the plaque was determined by nested polymerase chain reaction (PCR) . Antichlamydial immunoglobulin G (IgG), A (IgA) and M (IgM) was examined by microimmunofluorescence and compared to the PCR result . FINDINGS: DNA of C . pneumoniae was detected in 57 (75%) of 76 atherosclerotic lesions . In most cases (74/76: 97%) a positive IgA, IgM or IgG result was seen . Seven (12%) and 54 (94%) of the 57 PCR positive plaques came from patients with a positive IgM and IgA result, respectively . There was no statistical significant difference between PCR positive and PCR negative plaques in patients with a positive or negative serological result . Clinical characteristics were similarly distributed in patients with and without infected lesions . INTERPRETATION: C . pneumoniae organisms are frequently found in the atherosclerotic lesions of patients undergoing coronary surgery for unstable angina . Neither serological results of acute or recent infection by C . pneumoniae nor clinical characteristics are useful in predicting the individual risk of harbouring C . pneumoniae in the coronary lesions of patients with unstable angina.

Antiviral Res, 2003 May, 58(3), 209 - 15
Lactoferrin and lactoferricin inhibit Herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir; Andersen JH et al.; Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses . Upon peptic digestion of LF, a peptide called lactoferricin (Lfcin) is generated . Lfcin corresponds to the N-terminal part of the protein . In this study we investigated the antiviral activity of bovine and human Lfcin against Herpes simplex virus (HSV)-1 and HSV-2 . The 50% effective concentrations (EC(50)) for LF and Lfcin against several clinical isolates of HSV-1 and HSV-2, including acyclovir (ACV)-resistant strains, were determined . We further evaluated the effect of the combination of either LF or Lfcin with ACV against HSV-1 and HSV-2 . Synergy was observed between both LF or Lfcin in combination with ACV against the HSV laboratory strains.The 50% effective concentration (EC(50)) for ACV and LF or Lfcin, when combined with ACV, could be reduced by two- to sevenfold compared to the EC(50) when the drugs were used alone.

Trends Biochem Sci, 2003 May, 28(5), 234 - 7
The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases; Bateman A et al.; Cleavage of peptidoglycan plays an important role in bacterial cell division, cell growth and cell lysis . Here, we reveal that several known peptidoglycan amidases fall into a family, which includes many proteins of previously unknown function . The family includes two different peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity . The family includes the amidase portion of the bifunctional glutathionylspermidine synthase/amidase enzyme from bacteria and pathogenic trypanosomes . The glutathionylspermidine synthase is thought to be a key component of the alternative pathway in trypanosomes for protection from oxygen-radical damage and has been proposed as a potential drug target . The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain is often found in association with other domains that cleave peptidoglycan . The large number of multifunctional hydrolases suggests that they might act in a cooperative manner to cleave specialized substrates.

Trends Biochem Sci, 2003 May, 28(5), 226 - 9
The STIR-domain superfamily in signal transduction, development and immunity; Novatchkova M et al.; We have identified a conserved sequence segment in transmembrane receptors (including SEFs, IL17Rs) and soluble factors (including CIKS/ACT1) in eukaryotes and bacteria - the SEFIR domain . This sequence domain is part of the new STIR domain superfamily comprising also the TIR domain known to mediate TIR-TIR homotypic interactions . In TOLL/IL1R-like pathways, the cytoplasmically localized TIR domain of a receptor and the TIR domain of a soluble adaptor interact physically and activate signalling . The similarity between the SEFIR and TIR domains involves the conserved boxes 1 and 2 of the TIR domain that are implicated in homotypic dimerization, but there is no sequence similarity between SEFIR domains and the TIR sequence box 3 . By analogy, we suggest that SEFIR-domain proteins function as signalling components of Toll/IL-1R-similar pathways and that their SEFIR domain mediates physical protein-protein interactions between pathway components.

Biochim Biophys Acta, 2003 Jun 5, 1604(2), 77 - 94
Intrinsic and extrinsic uncoupling of oxidative phosphorylation; Kadenbach B; This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane . The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described . Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H(+)/e(-) stoichiometry=slip) is reviewed . Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping . Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential DeltaPsi, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate . In addition, two mechanisms of respiratory control are described . The first occurs through the membrane potential DeltaPsi and maintains high DeltaPsi values (150-200 mV) . The second occurs only in mitochondria, is suggested to keep DeltaPsi at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation . Finally, the regulation of DeltaPsi and the production of reactive oxygen species (ROS) in mitochondria at high DeltaPsi values (150-200 mV) are discussed.

Bioresour Technol, 2003 Apr, 87(2), 137 - 46
Polyhydroxyalkanoates: an overview; Reddy CS et al.; Polyhydroxyalkanoates have gained major importance due to their structural diversity and close analogy to plastics . These are gaining more and more importance world over . Different sources (natural isolates, recombinant bacteria, plants) and other methods are being investigated to exert more control over the quality, quantity and economics of poly(3-hydroxybutyrate) (PHB) production . Their biodegradability makes them extremely desirable substitutes for synthetic plastics . The PHB biosynthetic genes phbA, phbB and phbC are clustered and organized in one phbCAB operon . The PHB pathway is highly divergent in the bacterial genera with regard to orientation and clustering of genes involved . Inspite of this the enzymes display a high degree of sequence conservation . But how similar are the mechanisms of regulation of these divergent operons is as yet unknown . Structural studies will further improve our understanding of the mechanism of action of these enzymes and aid us in improving and selecting better candidates for increased production . Metabolic engineering thereafter promises to bring a feasible solution for the production of "green plastic".

Lupus, 2003, 12(5), 413 - 7
Atypical cutaneous findings in a patient with systemic lupus erythematosus; Elkayam O et al.; A previously diagnosed systemic lupus erythematosus patient presented with arthralgia, skin rash and muscular weakness . When treated with high-dose corticosteroids and methotrexate she improved, except for a persistent lesion in the hand which evolved into a profound ulcer, along with tender subcutaneous nodules in the calf . A skin biopsy disclosed necrotizing vasculitis with giant cell granuloma revealing acid fast positive bacteria on ziels nilsen staining . A chest X-ray disclosed miliary tuberculosis (TB) . The patient was diagnosed as miliary TB with prominent cutaneous involvement and treated with four anti-tuberculous drugs with slow resolution of her systemic, pulmonary and skin signs.

Mund Kiefer Gesichtschir, 2003 May, 7(3), 146 - 50 Epub 2003 Apr 10.
{James Israel (1848-1926) . His contribution to oral and maxillofacial surgery}; Knoner W et al.; The Berlin surgeon James Israel (1848-1926) is generally known as one of the leading kidney surgeons of his time . Furthermore, he is commemorated for describing actinomycosis bacteria, named after him in 1878: Actinomyces israelii . It is less well known that he also made several important contributions to maxillofacial surgery . This special aspect as well as Israel's biography is presented in this article from the history of medicine.

J Biol Inorg Chem, 2003 May, 8(5), 540 - 8 Epub 2003 Feb 15.
Reduced hybrid cluster proteins (HCP) from Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio vulgaris (Hildenborough): X-ray structures at high resolution using synchrotron radiation; Aragao D et al.; The hybrid cluster proteins from the sulfate reducing bacteria Desulfovibrio desulfuricans ATCC 27774 ( Dd) and Desulfovibrio vulgaris strain Hildenborough ( Dv) have been isolated and crystallized anaerobically . In each case, the protein has been reduced with dithionite and the crystal structure of the reduced form elucidated using X-ray synchrotron radiation techniques at 1.25 A and 1.55 A resolution for Dd and Dv, respectively . Although the overall structures of the proteins are unchanged upon reduction, there are significant changes at the hybrid cluster centres . These include significant movements in the position of the iron atom linked to the persulfide moiety in the oxidized as-isolated proteins and the sulfur atom of the persulfide itself . The nature of these changes is described and the implications with respect to the function of hybrid cluster proteins are discussed.

ALTEX, 2003, 20(2), 77 - 84
Proof of principle: detection of genotoxicity by a fluorescence-based recombination test in mammalian cells; Akyuz N et al.; Genotoxicity tests available today have several shortcomings . The widely applied Ames assay measures mutations in bacteria, thereby disregarding the physiological particularities of the human cell and organism.We provide first evidence for a new concept of genotoxicity detection in living human cell cultures . The data were obtained by use of a newly developed assay, which is based on the quantification of fluorescent signals, i.e . counting of the relative number of fluorescent cells in the sample . It is characterised by a short reaction time and fulfills the requirements for automated performance . The new system monitors chromosomal rearrangements and, therefore, is predicted to detect a broad spectrum of genotoxic substances . Indeed, we demonstrate the genotoxic effect of ionising radiation, of an Ames assay positive compound, and of two compounds which are poorly mutagenic in the Ames assay . The new assay will be optimised further and adapted to the requirements for routine analysis in order to help to further reduce animal experimentation in genotoxicity testing.

Mem Inst Oswaldo Cruz, 2003 Mar, 98(2), 265 - 8 Epub 2003 May 15.
Use of polymerase chain reaction and enzymatic cleavage in the identification of Helicobacter spp . in gastric mucosa of human beings from North Paraná, Brazil; Camargo PL et al.; Helicobacter pylori is the most common gastric bacteria of human beings . Animal-borne helicobacter have been associated with gastritis, ulceration, and gastric mucosa-associated lymphoid-tissue lymphoma in people . We attempted to identify the species of Helicobacter spp . that infect human beings in north Paran , Brazil . Samples of gastric mucosa from 38 dyspeptic patients were analyzed by optic microscopy on silver stained slides, polimerase chain reaction (PCR), and enzymatic cleavage . Genus and species-specific primers to H . pylori, H . heilmannii, H . felis, and consensual primers to H . bizzozeronii or H . salomonis were used . The PCR products were submitted to enzymatic cleavage by VspI (Helicobacter spp . product) and HinfI (species products) enzymes . Thirty-two out of 38 patients evaluated had 3.2 to 5 m long bacteria that resembled H . pylori in Warthin-Starry stained slides and were positive to the genus Helicobacter by PCR . In 30 of these patients the bacteria were identified as H . pylori . Two samples positive by silver stain were negative to all species tested by PCR . None of the 38 samples was positive to animal-origin helicobacter species . These results show that PCR and enzymatic restriction are practical methods to identify the species of helicobacters present in gastric mucosa of human beings . People in north Paran appear to be infected mostly with H . pylori.

Vaccine, 2003 Jun 1, 21 Suppl 2, S38 - 42
Human natural killer cell function and their interactions with dendritic cells; Moretta L et al.; Natural killer (NK) cells have long been considered as "primitive" and "non-specific" effector cells . However, the past 10 years have witnessed dramatic progress in our understanding of how NK cells function and their role in innate defenses . Thanks to specialized inhibitory receptors specific for MHC-class I molecules, they can sense the decrease or loss of these molecules, a typical condition of potentially dangerous cells such as tumor or virally-infected cells . NK cell triggering and lysis of these cells is mediated by several activating receptors and co-receptors that have recently been identified and cloned . While normal cells are usually resistant to the NK-mediated attack, a remarkable exception is represented by dendritic cells (DC) . In their immature form (iDC), they are susceptible to NK-mediated lysis because of the expression of low levels of surface MHC-class I molecules . Since the process of DC maturation (mDC) is characterized by the surface expression of high levels of MHC-class I molecules, mDC become resistant to NK cells . Exposure to live bacteria induces rapid DC maturation and, thus, resistance to NK cells . The cross-talk between DC and NK cells is more complex and involves also a DC-dependent NK cell activation and proliferation . Thus, two important players of the innate immunity may be involved in a coordinated regulation of critical events occurring at the interface between innate and adaptive immunity.

Best Pract Res Clin Gastroenterol, 2003 Jun, 17(3), 369 - 81
Gastrointestinal disorders of the critically ill . Shock liver; Strassburg CP; Shock liver describes a collecting pool of critically ill patients in whom the elevation of liver function tests or overt hepatic dysfunction is apparent . Different grades of shock liver affect about 50% of all intensive-care patients, varying from a mild elevation of serum aminotransferase and bilirubin levels in septic patients to an acute onset of high serum aminotransferases after haemodynamic shock . Abnormalities can subside within days or progressively deteriorate when persistent hepatic microcirculatory failure is present . Although hepatic injury in critically ill patients influences mortality rates it is underdiagnosed . The underlying pathophysiology involves changes in the portal and arterial blood supply as well as in microcirculation . Cross-talk between hepatocytes, Kupffer cells and endothelial cells, leading to an inflammatory response mediated primarily by tumour necrosis factor-alpha (TNF-alpha), is central to shock liver . The liver is a victim of shock inducers, and can also be the orchestrator of the inflammatory response syndrome (IRS) . Hepatic injury by TNF-alpha is modulated by the prevalent pro-inflammatory or anti-inflammatory mediator profile elaborated by Kupffer cells . Kupffer cells additionally participate in the clearance of endotoxin, bacteria and inflammatory mediators and are thereby capable of preventing IRS . The hepatocyte undergoes dramatic alterations in synthetic activity, biliary transport, bile flow and glucose metabolism . Although standard determinations of aminotransferases, coagulation studies, glucose, lactate and bilirubin can detect hepatic injury they only partially reflect the cellular mechanisms driving shock liver . The management of shock liver is focused on the prevention of precipitating causes by controlling sepsis, circulation parameters and metabolism in addition to the cautious monitoring of therapeutic measures that can increase hepatic injury, which include intravenous nutrition, mechanical ventilation and catecholamine administration.

Biochem Biophys Res Commun, 2003 Jun 6, 305(3), 641 - 8
Only one dnaK homolog, dnaK2, is active transcriptionally and is essential in Synechocystis; Varvasovszki V et al.; We examined the expression and the function of the DnaK chaperone family in the photoautotrophic cyanobacterium, Synechocystis PCC 6803 . Surprisingly, only one of the three dnaK genes was transcribed either under normal or heat shock conditions . Their predicted cochaperones (four dnaJs and one grpE) proved to be uninducible under our experimental conditions . Attempts to inactivate the active dnaK2 has failed, indicating that the gene is essential . The partial mutant displayed lower inducibility of chaperones (especially GroEL and HSP17) both at mRNA and protein levels upon heat shock . The mutant showed temperature sensitive phenotype, but was able to acquire thermotolerance.

Org Lett, 2003 May 29, 5(11), 1875 - 8
Stereoselective synthesis of functionalized precursors of the CDEF and CDE 2,6-dideoxy-tetra- and trisaccharide units of durhamycins A and B; Durham TB et al.; {structure: see text} Highly stereoselective syntheses of functionalized precursors of the CDEF and CDE 2,6-dideoxy-tetra- and trisaccharide units of the anti-HIV aureolic acids durhamycins A and B using 2-deoxy-2-iodo- and 2-deoxy-2-bromopyranosyl donors are described.

J Submicrosc Cytol Pathol, 2003 Jan, 35(1), 91 - 7
Bacteriocyte population growth in Blattella germanica; Lambiase S et al.; The number of bacteriocytes with nucleus in the M or S phase was analysed in relation to their rate of increase, throughout two nymphal instars (N1 and N6) of Blattella germanica Linnaeus (Blattaria, Blattellidae) . We treated the experimental specimens with colcemid in order to visualize C-metaphases, and with labelled thymidine in order to evidence DNA synthesis in these bacteria-carrying cells . In both young and old nymphs, the C-metaphases showed a similar trend: their average number was only 0.3 per 100 bacteriocytes counted throughout the entire instars . In the young nymphs, the number of bacteriocytes in the S phase was congruent with the frequencies of the C metaphases . Since the number of M or S bacteriocytes was not sufficient to account for the observed increase in number of these endosymbiotic cells during nymphal development, we hypothesize a mechanism of bacterial transmission from bacteriocytes to other fat body cells that may explain the numerical growth of the bacteriocyte population.

J Biochem (Tokyo), 2003 Jan, 133(1), 59 - 66
Development and characterization of a novel fusion protein composed of a human IgG1 heavy chain constant region and a single-chain fragment variable antibody against Venezuelan equine encephalitis virus; Hu WG et al.; Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus . It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy . In order to minimize the immunogenicity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed . It encoded a human IgG1 heavy chain constant region and a single-chain fragment variable antibody of 1A4A1 . After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro . The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recognition by protein G and human complement C1q binding . On non-reducing and reducing gel electrophoresis analysis of proteolytic fragments of the recombinant antibody, disulfide bond formation was found in the hinge region of the antibody . From these data, it was concluded that the recombinant antibody was capable of antigen recognition, and retained several functional activities . This work forms the basis for characterization of the recombinant antibody as to efficacy in vivo.

Infect Immun, 2003 Jun, 71(6), 3667 - 72
Natural history of Helicobacter hepaticus infection in conventional A/J mice, with special reference to liver involvement; Avenaud P et al.; It has been reported that Helicobacter hepaticus infection of mice leads to chronic hepatitis and hepatocarcinoma . Our aim was to monitor a cohort of 80 conventional A/J mice in which half of the mice were infected by H . hepaticus in order to study the evolution of the infection and the pathological changes in comparison to uninfected mice . H . hepaticus was detected by culture only in some colon and cecum specimens after 17 months of age, while PCR detected H . hepaticus in the intestines of all inoculated mice after only 5 months of infection . The percentage of mice in which H . hepaticus was detected in the gallbladder, bile ducts, and liver by PCR, as well as the number of bacteria present in the liver, tended to increase with increasing age and longer infection time . Anti-H . hepaticus immunoglobulin G antibodies were positive by enzyme-linked immunosorbent assay only in inoculated mice . Pathological findings were also more frequent as the mice grew older: fibrosis was present (especially in the peripheral part of the liver), and significant portal inflammation including lymphoid nodules was present in almost all infected animals . Biliary lesions of neutrophilic acute cholangitis or lymphocytic cholangitis were noted . However, lesions were also observed in uninfected animals, although at a significantly lower level, and the only hepatocellular carcinoma occurred in an uninfected mouse . The evolution towards hepatocarcinoma is not always the endpoint and may depend on the bacterial strain and on the environmental conditions.

Infect Immun, 2003 Jun, 71(6), 3597 - 602
Comprehensive analysis of the factor h binding capabilities of borrelia species associated with lyme disease: delineation of two distinct classes of factor h binding proteins; McDowell JV et al.; Some Lyme disease spirochete isolates can bind complement regulatory protein factor H (fH), a process that may allow evasion of complement-mediated killing . Here we demonstrate significant differences in the fH binding capabilities of species of the Borrelia burgdorferi sensu lato complex . The percentages of B . burgdorferi, B . afzelii, and B . garinii bacteria that bound fH in either enzyme-linked immunosorbent assays or affinity ligand binding immunoblot assays were 100, 83, and 29%, respectively . The fH binding protein profiles were examined and found to exhibit variability among isolates and to form two distinct classes . Differences in fH binding ability may contribute to the differences in pathogenesis and clinical course observed upon infection with different species of the B . burgdorferi sensu lato complex.

Infect Immun, 2003 Jun, 71(6), 3320 - 8
Association of Actinobacillus pleuropneumoniae capsular polysaccharide with virulence in pigs; Bandara AB et al.; The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine . However, a molecular investigation of whether the type or quantity of CP affects A . pleuropneumoniae virulence has not been reported . To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A . pleuropneumoniae serotype 1 was cloned and sequenced . Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes . A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector . The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively . Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP . The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively . Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074 . Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074 . With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B . Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55) . However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain . Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A . pleuropneumoniae correlated with the virulence of the bacteria in pigs . However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Zhonghua Kou Qiang Yi Xue Za Zhi, 2003 Jan, 38(1), 27 - 30
{Cloning and polymorphism analysis of prtH gene from Porphyromonas gingivalis}; Zheng Y et al.; OBJECTIVE: To clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis . METHODS: Using PCR, the prtH was amplified and cloned into pGEM-T vector . To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe . RESULTS: Recombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay . Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns . Strain 47A-1 seemed even not to contain prtH gene . CONCLUSIONS: Different prtH gene sequences exist in different P.g strains . This polymorphism may indicate various potential virulent effects during the infection and pathogenesis . Established PCR protocol is sensitive for identification of prtH gene.

Rev Esp Enferm Dig, 2003 Feb, 95(2), 97 - 104, 89-96
Helicobacter pylori infection affects the expression of PCNA, p53, c-erbB-2 and Bcl-2 in the human gastric mucosa; Jorge O et al.; OBJECTIVE: Helicobacter pylori infection has been related to gastric carcinogenesis . This association is based on epidemiological data, pathological changes observed in the gastric mucosa, and chemical products from bacteria that may induce damage of DNA . In the present study we examined gastric endoscopic biopsies from patients with chronic gastritis, with and without H . pylori infection, and surgical biopsies from gastric cancer patients to evaluate whether this bacteria may induce changes in the expression of molecular markers associated with carcinogenesis . PATIENTS AND METHODS: the study involved 57 biopsies from the antral region of the stomach of patients with chronic gastritis and gastric cancer that were analyzed by immunohistochemistry . Molecular markers examined were: PCNA (Proliferating Cell Nuclear Antigen), p53, c-erbB-2, Bcl-2, and p21 H-ras . RESULTS: PCNA content of epithelial cells was significantly higher in H . pylori infected biopsies . Treatment aimed to eradicate H . pylori decreased the level of PCNA-positive cells in the group of patients that became H . pylori-negative as well as in H . pylori-positive patients . Nuclear p53 expression (used here as a surrogate marker for p53 mutation/inactivation) and c-erbB-2 expression were observed only in the group of patients that remained with the bacteria after treatment . A higher bcl-2 expression in lymphoid cells was observed in H . pylori-positive biopsies, and treatment did not change the expression of this protein . No significant expression of p21 H-ras was observed in the studied biopsies . CONCLUSION: this study suggests that H . pylori is involved in the induction of molecular changes that might predispose human gastric mucosa cells to pre-neoplastic and neoplastic events.

J Immunol, 2003 Jun 1, 170(11), 5607 - 14
Subversion and utilization of the host cell cyclic adenosine 5'-monophosphate/protein kinase A pathway by Brucella during macrophage infection; Gross A et al.; Brucella spp . are intramacrophage pathogens that induce chronic infections in a wide range of mammals, including domestic animals and humans . Therefore, the macrophage response to infection has important consequences for both the survival of phagocytosed bacteria and the further development of host immunity . However, very little is known about the macrophage cell signaling pathways initiated upon infection and the virulence strategy that Brucella use to counteract these responses and secure their survival . In a previous study, we have shown that macrophages activated by SR141716A, a ligand of the cannabinoid receptor CB1, acquired the capacity to control Brucella and observed that the CB1 receptor-triggering engages the microbicidal activity of phagocytes . To analyze the perturbation of cell signaling pathway during macrophage infection by Brucella, we hypothesized that SR141716A provides cell signaling that interferes with the bacterial message leading to inhibition of macrophage functions . As CB1 receptor belongs to the family of G protein-linked receptors, we explored the cAMP signaling pathway . In this study, we show that the CB1 ligand inhibited the bacteria-induced cell signaling . Taking advantage of this result, we then demonstrated that Brucella infection elicited a rapid activation of the cAMP/protein kinase A pathway . This activation resulted in a prolonged phosphorylation of the transcription factor CREB . We finally demonstrate that the activation of the cAMP/protein kinase A pathway is crucial for the survival and establishment of Brucella within macrophages . For the first time in phagocytes, we thus characterized a primordial virulence strategy of Brucella involving the host signaling pathway, a novel point of immune intervention of this virulent pathogen.

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