J Chromatogr B Analyt Technol Biomed Life Sci, 2005 Feb 5, 815(1-2), 251 - 60 Proteomics and physiology of erythritol-producing strains; Park YC et al.; In-depth knowledge bases on physiological properties of microbes are required to design a better microbial system at a gene level and to develop an industrially viable process in an optimized scheme . Proteomic analyses of industrially useful microorganisms are particularly important for achieving such objectives . In this review, industrial application of erythritol in food and pharmaceutical areas and proteomic techniques for erythritol-producing microbes were presented . Proteomic technologies for erythritol-producing strains such as Candida magnoliae contained protein or peptide sample preparation for two-dimensional electrophoresis and mass spectrometry, analysis of proteome with matrix assisted laser desorption-ionization/time-of-flight mass spectrometry, liquid chromatography/electrospray ionization/tandem mass spectrometry and similarity searching algorithms . The proteomic information was applied to predict the carbon metabolism of erythritol-synthesizing microorganisms. J Antimicrob Chemother . 2005 Jan 13; {Epub ahead of print} A review of the microbiology, antibiotic usage and resistance in chronic skin wounds; Howell-Jones RS et al.; Chronic leg and foot wounds represent an increasing burden to healthcare systems as the age of the population increases . The deep dermal tissues of all chronic wounds harbour microorganisms, however, the precise interaction between microbes in the wounds and impaired healing is unknown . With regard to antibiotic therapy, there is a lack of evidence concerning its effectiveness, optimal regimens or clinical indications for treatment . Despite this lack of evidence, antibiotics are frequently a feature of the management of chronic wounds and these patients receive significantly more antibiotic prescriptions (both systemic and topical) than age and sex-matched patients . Current guidelines for antibiotic prescribing for such wounds are often based on expert opinion rather than scientific fact and may present difficulties in interpretation and implementation to the clinician . Although the increasing prevalence of antibiotic resistance is widely recognized, the relationships between antibiotic resistance, chronic wound microbiology and rationales for antibiotic therapy have yet to be determined . This review discusses the role of microbes in chronic wounds from a clinical perspective with particular focus on the occurrence of bacteria and their impact on such wounds . The evidence and role of antibiotics in the treatment of such wounds are outlined and current practice of antibiotic usage for chronic wounds in the primary care setting described . The implications of antibiotic usage with regard to antibiotic resistance are also considered. Compend Contin Educ Dent, 2004 Sep, 25(9 Suppl 1), 4 - 8 Why is there and should there be more attention paid to dental erosion? Hefferren JJ. Fluoride delivered by fluoridation, toothpaste, and mouth rinses, combined with regular preventive professional and personal care, has given us a much better opportunity to keep our teeth for a lifetime . But in contrast to better preventive care, lifestyle changes leading to stress or dietary changes in what and how we drink and eat may adversely affect the esthetics and/or healthy look of our teeth . Surface-enamel wear can be described as the result as well as a chronicle of an individual's life . Components of wear--including abfraction, attrition, abrasion, and erosion--combine to characterize the lifetime of wear . The chemical-erosive-wear component appears to be changing because of modern cultural life choices . A number of literature reports conclude that excessive exposure to various acid sources can contribute to the chemical erosion of tooth surfaces . For example, the hydrochloric acid regurgitated from the stomach, the acid added to swimming pools to combat microbes, the citric and chelating carboxylic acids of fruit and fruit-based beverages, the phosphoric acid of cola beverages, and the acetic acid of vinegars used for salads all can contribute to the chemical erosion of tooth surfaces . While these acid sources have the potential to erode, our saliva and the manner in which we consume these substances can largely modify the erosive action of the acids they contain . With a better understanding of our personal lifestyle choices coupled with the educated guidance of our profession, we can help to reduce the opportunities for erosive wear to occur. J Cosmet Sci, 2004, 55 Suppl, S1 - S17 Surfactants, polymers and their nanoparticles for personal care applications; Somasundaran P et al.; A "touch me not" plant folding up rapidly upon being attacked or microbes depositing on teeth or ocean vessels even under hostile conditions are examples in nature that provide inspiration for developing new classes of personal care release or deposition systems . In this paper, development of such systems based on polymer/surfactant colloid chemistry is explored for achieving transport and release of cosmetic and pharmaceutical molecules at desired rates at desired sites . The successful development of products depends upon understanding and utilizing key interactions among surfactants, polymers and hybrid polymers that are relevant to personal care products . Thus, the absorbed layers or tethers on the particulates can be manipulated for desired dispersion of actives or depositions on substrate under any and all conditions . New hybrid polymers and nanogels have been synthesized for tuning up nanodomains that can extract and deliver at will cosmetics/drugs/toxins by perturbing pH, temperature or ionic strength of the system . Particularly, hydrophobically modified polymers have features of both polymers and surfactants and due to the associative nature of the hydrophobic groups, such polymers can form intramolecular nanodomains for performing carrier functions . Nanogels developed recently include that of polyacrylamide, poly(acrylic acid) and starch nanogels modified for extraction and subsequent slow release of fragrances and overdosed toxic drugs . Binding and release processes were investigated using surface plasmon resonance and fluorescence spectroscopies, powerful techniques for monitoring short term and long term changes. Can J Microbiol, 2004 Oct, 50(10), 793 - 802 Microcosm tests of the effects of temperature and microbial species number on the decomposition of Carex aquatilis and Sphagnum fuscum litter from southern boreal peatlands; Thormann MN et al.; Increased decomposition rates in boreal peatlands with global warming might increase the release of atmospheric greenhouse gases, thereby producing a positive feedback to global warming . How temperature influences microbial decomposers is unclear . We measured in vitro rates of decomposition of senesced sedge leaves and rhizomes (Carex aquatilis), from a fen, and peat moss (Sphagnum fuscum), from a bog, at 14 and 20 degrees C by the three most frequently isolated fungi and bacteria from these materials . Decomposition rates of the bog litter decreased (5- to 17-fold) with elevated temperatures, and decomposition of the sedge litters was either enhanced (2- to 30-fold) or remained unaffected by elevated temperatures . The increased temperature regime always favoured fungal over bacterial decomposition rates (2- to 3-fold) . Different physiological characteristics of these microbes suggest that fungi using polyphenolic polymers as a carbon source cause greater mass losses of these litters . Litter quality exerted a stronger influence on decomposition at elevated temperatures, as litter rich in nutrients decomposed more quickly than litter poorer in nutrients at higher temperatures (8.0%&ndash;25.7% for the sedge litters vs . 0.2% for the bryophyte litter) . We conclude that not all peatlands may provide a positive feedback to global warming . Cautious extrapolation of our data to the ecosystem level suggests that decomposition rates in fens may increase and those in bogs may decrease under a global warming scenario. Annu Rev Plant Biol . 2004 Nov 3; {Epub ahead of print} Phytoremediation; Pilon-Smits E; Phytoremediation, the use of plants and their associated microbes for environmental cleanup, has gained acceptance in the past 10 years as a cost-effective, noninvasive alternative or complementary technology for engineering-based remediation methods . Plants can be used for pollutant stabilization, extraction, degradation, or volatilization . These different phytoremediation technologies are reviewed here, including their applicability for various organic and inorganic pollutants, and most suitable plant species . To further enhance the efficiency of phytoremediation, there is a need for better knowledge of the processes that affect pollutant availability, rhizosphere processes, pollutant uptake, translocation, chelation, degradation, and volatilization . For each of these processes I review what is known so far for inorganic and organic pollutants, the remaining gaps in our knowledge, and the practical implications for designing phytoremediation strategies . Transgenic approaches to enhance these processes are also reviewed and discussed . Expected online publication date for the Annual Review of Plant Biology Volume 56 is April 28, 2005 . Please see for revised estimates. Oncol Rep, 2005 Feb, 13(2), 179 - 84 Association of disease progression and poor overall survival with detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer; Bauernhofer T et al.; The aim of this study was to define the frequency and clinical relevance of cytokeratin positive metastatic tumor cells in the peripheral circulation of patients with stage IV breast cancer . Peripheral blood was collected from 32 consecutive patients with metastatic breast cancer and 23 healthy donors . Tumor cells were enriched using positive selection with anti-HEA125-microbeads and cytospins were prepared of the positive selection eluate . Slides were incubated with a Fab(2) fragment of the pancytokeratin antibody A45-B/B3 conjugated with alkaline phosphatase (AKP) and a CAM5.2-AKP monoclonal antibody and developed with an alkaline phosphatase anti-alkaline phosphate reaction (APAAP) . All samples were evaluated using light microscopy and an automated image analysis system . In 8/32 (25%) patients cytokeratin positive (CK(+)) cells could be detected after anti-HEA125 enrichment in the peripheral blood whereas in none out of 23 healthy donors . One to 1000 (median 5) positive cells per patient sample were observed and cluster of tumor cells in one patient . Automated image analysis was as powerful in detecting micrometastases as conventional light microscopy . All patients with CK(+) cells in the peripheral circulation (8/8, 100%) showed progressive disease at the time-point of blood draw whilst only 9/24 (37.5%) showed disease progression without detection of positive cells . The median overall survival of CK(+) patients was 4+/-2 months compared to 13+/-7 months of CK(-) patients (p<0.001) . CK(+) cells are detectable in the peripheral circulation of 25% of patients with metastatic breast cancer after positive selection with anti-HEA125 . Detection of tumor cells in the peripheral circulation might be correlated with progression of disease and shorter overall survival. Genome Biol . 2005;6(1):R5 . Epub 2004 Dec 23. Wound healing and inflammation genes revealed by array analysis of 'macrophageless' PU.1 null mice; Cooper L et al.; BACKGROUND: Wound healing is a complex process requiring the collaborative efforts of different tissues and cell lineages, and involving the coordinated interplay of several phases of proliferation, migration, matrix synthesis and contraction . Tissue damage also triggers a robust influx of inflammatory leukocytes to the wound site that play key roles in clearing the wound of invading microbes but also release signals that may be detrimental to repair and lead to fibrosis . RESULTS: To better define key cellular events pivotal for tissue repair yet independent of inflammation we have used a microarray approach to determine a portfolio of over 1,000 genes expressed across the repair response in a wild-type neonatal mouse versus its PU.1 null sib . The PU.1 null mouse is genetically incapable of raising the standard inflammatory response, because it lacks macrophages and functioning neutrophils, yet repairs skin wounds rapidly and with reduced fibrosis . Conversely, by subtraction, we have determined genes that are either expressed by leukocytes, or upregulated by fibroblasts, endothelial cells, muscle cells and others at the wound site, as a consequence of inflammation . To determine the spatial expression pattern for several genes in each cluster we have also performed in situ hybridization studies . CONCLUSIONS: Cluster analysis of genes expressed after wounding wild-type mice versus PU.1 null sibs distinguishes between tissue repair genes and genes associated with inflammation and its consequences . Our data reveal and classify several pools of genes, giving insight into their likely functions during repair and hinting at potential therapeutic targets. Evol Dev, 2005 Jan-Feb, 7(1), 3 - 17 Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution; Kuraku S et al.; Summary The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified . In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace . The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning . To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft-shelled turtle, Pelodiscus sinensis, using microbead-based differential cDNA analysis and real-time reverse transcription-polymerase chain reaction . We identified orthologs of Sp5, cellular retinoic acid-binding protein-I (CRABP-I), adenomatous polyposis coli down-regulated 1 (APCDD1), and lymphoid enhancer-binding factor-1 (LEF-1) . Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage . In association with the expression of LEF-1, the nuclear localization of beta-catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development . These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co-option of pre-existing genes. Compend Contin Educ Dent, 2004 Jan, 25(1 Suppl), 24 - 9 Sterilization update 2003; Harte JA et al.; Instrument processing is a key part of the office infection control program . Each step in the process must be performed correctly to help ensure patient safety . The instrument processing area must be organized so that contaminated items are not confused with sterilized items, and so that sterilized items do not accidentally become recontaminated . Instruments need to be cleaned completely of visible debris using an ultrasonic cleaner or instrument washer . The cleaned instruments are packaged before sterilization to protect them from recontamination until they are opened for use for the next patient . Processing the packaged instruments through a heat sterilizer (steam, dry heat, or unsaturated chemical vapor) kills any microbes that remain on the instruments . The sterile packages are handled and stored in a manner that preserves the integrity of the packaging material . The use and functioning of the sterilizer is monitored by mechanical, chemical, and biological means, and records are kept to document these evaluations . Sterilization failures are addressed carefully so that patient safety can be maintained. Lab Invest . 2005 Jan 10; {Epub ahead of print} Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique; Morsy MA et al.; Intrahepatic lymphocytes (IHL) with their diverse and distinctive subsets emphasise the importance of the liver as a site of immunological activity, but special care is required for their isolation and characterisation . Protocols for IHL isolation, purification and FACS analysis were devised and compared with published extraction protocols . We have reduced the time that IHL are exposed to potentially damaging enzymes during extraction and purified specific subsets using monoclonal antibody (mAb)-coated magnetic microbeads . This has yielded IHL populations with higher viability than previously described protocols (92-95%, compared with 39-86%) . Flow cytometric characterisation of IHL subset immunophenotypes was optimised by combining CD45 staining (fluorescence gating) with traditional light scatter properties . Using a panel of mAb and liver biopsies obtained from 23 cadaveric liver transplant donors, we show that the normal liver contains a heterogeneous IHL population with distinctive phenotypes . CD8(+) IHL was the predominant population with a mean CD4/CD8 ratio of 1:1.7 . Up to 40% of IHL expressed gammadeltaTCR and a third expressed CD56 NK marker; indicating a site of intense immunological activity . The techniques described will allow these cell types to be isolated, fully characterised and their physiological functions to be determined . The histologically normal liver contains heterogeneous and diverse IHL with large numbers of CD8(+), NK, NKT and gammadelta(+) cells.Laboratory Investigation advance online publication, 27 December 2004; doi:10.1038/labinvest.3700219. APMIS, 2004 Dec, 112(11-12), 898 - 907 The development of tools for diagnosis of tularemia and typing of Francisella tularensis; Johansson A et al.; Johansson A, Forsman M, Sjostedt A . The development of tools for diagnosis of tularemia and typing of Francisella tularensis . APMIS 2004;112:00-00.Rapid development of molecular techniques for the diagnosis of infections and typing of microbes has been seen during the last 10 years . The present review exemplifies this development by presenting the work of the authors and others regarding techniques for the diagnosis of tularemia and typing of Francisella tularensis . The lack of rapid and safe methods for the laboratory diagnosis of tularemia was the rationale behind the development of methods for the direct detection of F . tularensis in clinical specimens . Today, detection by polymerase chain reaction has become an important adjunct to clinical decisions for the early diagnosis of tularemia . The elucidation of the epidemiology and epizootology of the disease has been hampered by the lack of suitable methods . During recent years several DNA-based methods that allow rapid identification of the four F . tularensis subspecies, including differentiation of strains of the two clinically important subspecies, the highly virulent type A strains and less virulent type B strains, have been developed . Since F . tularensis strains of any origin exhibit highly conserved genomic sequences, the availability of extensive genome sequence data was a prerequisite for the development of a typing system that allows discrimination of individual isolates . The most discriminatory method is based on multiple-locus variable-number tandem repeat analysis (MLVA) and uses highly variable parts of the F . tularensis genome . The method will be an important tool in future studies of the molecular epidemiology of tularemia. Curr Pharm Des, 2005, 11(1), 25 - 36 Mining the probiotic genome: advanced strategies, enhanced benefits, perceived obstacles; Callanan M; Recent advances in DNA sequencing has made it possible to accurately decipher the entire genetic complement of a probiotic bacterium . Increases in sequencing capabilities have been enhanced through improved computer software that can annotate, or identify, the majority of genes encoded by the sequence . The availability of annotated genome sequence will be important in defining the capabilities of the individual strains of probiotic bacteria . It will also form the platform for microarray and proteomic technologies that allow real-time analysis of RNA and protein expression in the bacterial cell . Investigation of probiotic organisms with these new and potentially powerful tools will facilitate the development of the bacteria as therapeutic agents, and provide the mechanisms to produce advanced probiotic strains . This paper addresses the core technologies in the rapidly growing area of genomics, and their application to the molecular characterisation of probiotic bacteria and host-microbe interactions. BMC Bioinformatics . 2005 Jan 6;6(1):3 {Epub ahead of print} Differences in codon bias cannot explain differences in translational power among microbes; Dethlefsen L et al.; BACKGROUND: Translational power is the cellular rate of protein synthesis normalized to the biomass invested in translational machinery . Published data suggest a previously unrecognized pattern: translational power is higher among rapidly growing microbes, and lower among slowly growing microbes . One factor known to affect translational power is biased use of synonymous codons . The correlation within an organism between expression level and degree of codon bias among genes of Escherichia coli and other bacteria capable of rapid growth is commonly attributed to selection for high translational power . Conversely, the absence of such a correlation in some slowly growing microbes has been interpreted as the absence of selection for translational power . Because codon bias caused by translational selection varies between rapidly growing and slowly growing microbes, we investigated whether observed differences in translational power among microbes could be explained entirely by differences in the degree of codon bias . Although the data are not available to estimate the effect of codon bias in other species, we developed an empirically-based mathematical model to compare the translation rate of E . coli to the translation rate of a hypothetical strain which differs from E . coli only by lacking codon bias . RESULTS: Our reanalysis of data from the scientific literature suggests that translational power can differ by a factor of 5 or more between E . coli and slowly growing microbial species . Using empirical codon-specific in vivo translation rates for 29 codons, and several scenarios for extrapolating from these data to estimates over all codons, we find that codon bias cannot account for more than a doubling of the translation rate in E . coli, even with unrealistic simplifying assumptions that exaggerate the effect of codon bias . With more realistic assumptions, our best estimate is that codon bias accelerates translation in E . coli by no more than 60% in comparison to microbes with very little codon bias . CONCLUSIONS: While codon bias confers a substantial benefit of faster translation and hence greater translational power, the magnitude of this effect is insufficient to explain observed differences in translational power among bacterial and archaeal species, particularly the differences between slowly growing and rapidly growing species . Hence, large differences in translational power suggest that the translational apparatus itself differs among microbes in ways that influence translational performance. J Biol Chem . 2005 Jan 5; {Epub ahead of print} Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by Mannan-binding protein a highly fucosylated polylactosamine type N-glycan; Terada M et al.; Mannan-binding protein (MBP) is a C-type serum lectin, and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine and fucose on microbes . In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice . We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) {Y . Ma et al., Proc . Natl . Acad . Sci . USA, 96, 371-375 (1999)} . In this study, the MBP-ligands on the surface of SW1116 cells were characterized . Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP-binding to SW1116 cells indicated that fucose plays a crucial role in the interaction . Subsequently, pronase glycopeptides were prepared from whole cell lysates and oligosaccharides were liberated by hydrazinolysis . After being tagged by pyridylamination, MBP-ligand oligosaccharides were isolated with an MBP-affinity column, and then their sequences were determined by MS and MS/MS after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation . The MBP-ligands were shown to be large, multi-antennary N-glycans carrying a highly fucosylated polylactosamine-type structure . At the non-reducing termini, Leb(b)-Lea(a) or tandem repeats of the Lea(a) structure prevail, a substantial proportion of which are attached via internal Lex(x) or N-acetyllactosamine units to the trimannosyl core . The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens . It is concluded that MBP requires clusters of tandem repeats of the Leb(b)/Lea(a) epitope for recognition. J Thromb Haemost, 2005 Jan, 3(1), 74 - 78 Autoimmune thrombocytopenia: flow cytometric determination of platelet-associated autoantibodies against platelet-specific receptors; Tomer A et al.; Summary . Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction . Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties . We here describe a feasible diagnostic method using the commonly available technique of flow cytometry . An antigen-specific assay for platelet-associated antibody was developed and tested in 62 adult patients with chronic ITP, 14 patients with thrombocytopenia of decreased production and 60 healthy controls . The method is based on flow cytometric (FCM) detection of autoantibodies reacting with specific platelet receptors immobilized on microbeads . The average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8-31.0), in the non-ITP thrombocytopenic patients 0.9 (range 0.7-1.2), and in the healthy controls 1.0 (range 0.7-1.3) . The average assay coefficient of variation was 0.218 {95% confidence interval (CI) 0.213, 0.221} . The difference between the ITP patients and both groups was highly significant (P < 0.001), using a stringent non-parametric analysis . A comparison of the FCM assay with the radioactive immunobead assay previously reported on the same cohort of patients showed significant correlation (R(2) = 0.71, 95% CI 0.39, 0.53) . The overall performance of the FCM assay in discriminating between ITP patients and normals was estimated by the receiver operating characteristic (ROC) plot, showing an area under the curve of 0.96 (maximal value 1.0), with standard error of 0.033 . We conclude that the present FCM assay is clinically useful for routine diagnosis and follow-up of ITP. Springer Semin Immunopathol . 2005 Jan 5; {Epub ahead of print} B cell superantigens: a microbe's answer to innate-like B cells and natural antibodies; Goodyear CS et al.; Marginal zone B cells and B-1 cells have been termed innate-like B cells as they express limited repertoires that play special roles in immune defenses against common infections . These B cells are the sources of natural antibodies and are capable of highly accelerated clonal responses that help counter blood-borne infections . We have characterized a class of microbial product with highly adapted binding interactions with host immunoglobulins/B cell receptors (BCRs), which enable the targeting of large supra-clonal sets of B cells for activation-associated apoptotic death . In recent studies, we have shown that all B cells with V region-targeted BCRs are susceptible . However, compared to follicular B cells, in vivo exposure preferentially causes innate-like B cells to undergo induced death with subsequent long-lasting supra-clonal depletion and immune tolerance . Based on these properties, it is likely that B cell superantigens influence the pathogenesis of some common infections, but also may provide novel therapeutic opportunities to treat B cell neoplastic and autoimmune diseases. Microsc Res Tech, 2004 Nov, 65(4-5), 218 - 225 Diffusion of microspheres in sealed and open microarrays; Rieger B et al.; In several experiments, we study the diffusion of microspheres with different radii in microarrays filled with a variety of aqueous solutions of ethylene glycol . We study diffusion in open and closed (sealed) microarrays . In sealed nanoliter wells, the tracers show pure diffusion, whereas in open reactors, a radial outward-directed evaporation-induced liquid flow is superimposed onto the diffusion . In general, one of the following quantities can be calculated if the others are known: the temperature, the viscosity of the medium, the radius of the microbeads, or the diffusion constant . The estimated diffusion constants in closed microarrays are in good agreement with theoretical predictions based on the Brownian motion . We monitor the motion of the microbeads under a microscope and extract their paths in time from the digital recordings . Ambiguous paths due to the crossing of two trajectories can be detected . We show that low microsphere concentrations or high viscosities do not hamper a robust estimation of the diffusion parameters . Microsc . Res . Tech . 65:218-225, 2004 . (c) 2005 Wiley-Liss, Inc. Trends Biotechnol, 2005 Jan, 23(1), 17 - 21 Transgenes for tea? Heritage J. So far, no compelling scientific evidence has been found to suggest that the consumption of transgenic or genetically modified (GM) plants by animals or humans is more likely to cause harm than is the consumption of their conventional counterparts . Despite this lack of scientific evidence, the economic prospects for GM plants are probably limited in the short term and there is public opposition to the technology . Now is a good time to address several issues concerning GM plants, including the potential for transgenes to migrate from GM plants to gut microbes or to animal or human tissues, the consequences of consuming GM crops, either as fresh plants or as silage, and the problems caused by current legislation on GM labelling and beyond. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 72 - 5 {Detection of IFN-gamma level in single CD8(+) T cell.}; Wang ZJ et al.; AIM:To establish a new method capillary electrophoretic immunoassay laser-induced fluorescence (CEIA-LIF), to detect IFN-gamma level in single CD8(+) T cell . METHODS: CD8(+) T cells were isolated from peripheral blood of two patients with severe aplastic anemia(SAA) and one normal person by Ficoll-Hypaque gradient centrifugation and then were purified by immunomagnetic microbead separation . Then purified CD8(+) T cells were incubated with digitonin for 15 min followed by FITC-anti-IFN-gamma mAb for 20 min . The single cell was detected continuously by CEIA-LIF . The feasibility of the method was confirmed by inverted microscopy and laser scanning confocal fluorescence microscopy . RESULTS: The IFN-gamma content in purified CD8(+) T cells was detected under the condition of cell-membrane integrity . The IFN-gamma level in single CD8(+) T cell from 2 SAA patients was (151.53+/-28.92)zmol and (223.72+/-45.23)zmol, respectively, and much higher that from normal control (47.47+/-17.97)zmol ( P=0.001) . CONCLUSION: It is feasible to quantitate IFN-gamma in single CD8(+) T cell by CEIA-LIF . CEIA-LIF might be useful in the clinical detection of intracellular cytokines. Biosens Bioelectron, 2005 Feb 15, 20(8), 1685 - 9 Sensitive bondforce measurements of ligand-receptor pairs with magnetic beads; Panhorst M et al.; The bondforces between biotinylated surfaces and streptavidin or avidin coated beads are investigated by a magnetic field based manipulation system for magnetic microbeads . The magnetic field is generated by currents through a set of conducting lines, and its gradient exerts a force onto the magnetic beads . The force can be increased until the bond between the bead and the surface breaks . Consistent with other groups we found two conformations for both investigated bonds . The measured bondforces for the two conformations are for Streptavidin-Biotin: 55.9 and 244.7 fN and for Avidin-Biotin: 15.9 and 58.4 fN . These very low bondforces (10-100 times smaller than earlier measurements) match to the extremely low loading rate of about 1 fN/s . This new technique thus allows to investigate biomolecular bonds by extremely low forces. Sci Total Environ, 2005 Jan 20, 337(1-3), 207 - 12 Shifts of soil enzyme activities in wetlands exposed to elevated CO(2); Kang H et al.; Wetlands play a key role in global biogeochemical cycles, and as such, the effects of global climatic changes on these systems are of great importance . In this study, we assessed impacts of elevated CO(2) on soil enzyme activities in different types of wetlands . We hypothesised that elevated CO(2), by increasing DOC supply into the soil, would modify enzyme activities . Intact soil cores collected from four wetlands (a bog, a fen, a gully mire, and a marsh) in north Wales and Korea were incubated either under ambient conditions (370 ppm) or elevated CO(2) conditions (double ambient) for 4-2 months . Elevated CO(2) increased DOC concentrations in the pore-water, by which soil microbes appeared to be affected . Enzyme activities exhibited various responses . For example, elevated CO(2) had no effect on beta-glucosidase activity in any soil, suggesting little direct impact on carbon mineralisation . However, N-acetylglucosaminidase activity increased significantly (P<0.05, n=5) in the cores from the bog, whilst a similar response was found in the gully mire for phosphatase activity . Such changes were absent from the fen and marsh where inorganic nutrients were abundant, suggesting that enzyme activities involved in N or P mineralisation only increase under elevated CO(2) when nutrient limitation is strongly exerted. Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 461 - 6 Epub 2004 Dec 29. From The Cover: Combinatorial biosynthesis of antitumor indolocarbazole compounds; Sanchez C et al.; Rebeccamycin and staurosporine are natural products with antitumor properties, which belong to the family of indolocarbazole alkaloids . An intense effort currently exists for the generation of indolocarbazole derivatives for the treatment of several diseases, including cancer and neurodegenerative disorders . Here, we report a biological process based on combinatorial biosynthesis for the production of indolocarbazole compounds (or their precursors) in engineered microorganisms as a complementary approach to chemical synthesis . We have dissected and reconstituted the entire biosynthetic pathway for rebeccamycin in a convenient actinomycete host, Streptomyces albus . This task was achieved by coexpressing different combinations of genes isolated from the rebeccamycin-producing microorganism . Also, a gene (staC) was identified in staurosporine-producing microbes and was shown to have a key role to differentiate the biosynthetic pathways for the two indolocarbazoles . Last, incorporation of the pyrH and thal genes, encoding halogenases from different microorganisms, resulted in production of derivatives with chlorine atoms at novel positions . We produced >30 different compounds by using the recombinant strains generated in this work. Radiat Res, 2004 Nov, 162(5), 585 - 91 Simulation of binary methods for the microdosimetric analysis of cell survival after alpha-particle irradiation: ability to distinguish between different models; Stinchcomb TG et al.; Analysis of cell survival after alpha-particle irradiation must account for the distribution in the amounts of energy deposited in each cell nucleus . Microdosimetric computations are usually used to determine these distributions . Irradiation with microbeams and other modern techniques has made these computations unnecessary for certain cell geometries . These techniques allow the survival of individual cells to be correlated with the amount of radiation delivered to individual cell nuclei . However, to maintain the individuality of data generated for each cell, new methods of analysis are required . In this study, we propose the use of binary methods . Each cell is regarded as a Bernoulli trial with a different probability for success (colony formation) . Parameter values of the survival model are chosen to maximize the likelihood of the observed outcome . To evaluate this method, simulated data for 500, 5000 and 50,000 cells irradiated by alpha particles are analyzed along with the associated outcome for four different cell survival models . Each survival model has a different dependence on the radius of the cell nucleus . These results indicate that the model that was simulated has the highest likelihood value in all cases . However, the ability to distinguish between competing models is present only for a larger numbers of cells. Autoimmunity, 2004 Sep, 37(5), 375 - 86 Myocarditis, microbes and autoimmunity; Whitton JL et al.; Acute and chronic myocarditis can be caused by a number of infectious agents, including viruses, bacteria and protozoa . These diseases are refractory to treatment, and the development of rational therapies will require a detailed understanding of the mechanisms that underlie the pathological inflammatory responses . Here, we review three infectious myocarditides that, despite the dissimilarity of the microorganisms, share several common features: (i) the microbes replicate in the heart; but (ii) are difficult to isolate, in infectious form, during chronic disease; (iii) autoreactive antibodies and T cells specific for cardiac antigens have been identified in infected animals; and (iv) these autoreactive responses have been proposed as the main effectors of cell death, and myocardial damage . We critically evaluate the data, and we suggest that the findings can be reconciled without invoking autoimmunity as an effector mechanism . Alternative hypotheses to explain the tissue destruction are proposed. Dev Cell, 2005 Jan, 8(1), 1 - 2 Guts and glory; balancing microbes and the immune response; Pal S et al.; Maintaining balance with intestinal flora is an important activity of the immune system in higher metazoans . In this issue of Developmental Cell, Ha et al . demonstrate a central role of a redox balance in microbial interactions in the fruit fly gut. Microb Pathog, 2004 Dec, 37(6), 295 - 302 Epub 2004 Dec 08. Expression of IL-6 and TNF-alpha in human alveolar epithelial cells is induced by invading, but not by adhering, Legionella pneumophila; Chang B et al.; Legionella pnemophila causes atypical pneumonia in humans, especially in patients with chronic pulmonary diseases and underlying immunosuppression, and in elderly people . Several previous studies have shown that L . pneumophila induced several inflammatory cytokines in murine macrophages, but little is known about cytokine induction by the bacterium in lung epithelial cells . In this study, we investigated the ability of L . pneumophila to stimulate the production of pro-inflammatory cytokines in the human A549 alveolar epithelial cell line during 24h exposure to 10(6), 10(7), and 10(8) microbes . Infection of the wild L . pneumophila strain to A549 resulted in increased levels of interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-alpha) mRNA, and also the secretion of their production into culture medium . In contrast, the level of mRNAs and proteins of IL-1beta and gamma interferon (IFN-gamma) remained unchanged and undetected, respectively . Production of IL-8, IL-6, and TNF-alpha in A549 decreased when an icmE multiplication-less mutant and the heat-killed L . pneumophila strain were inoculated . The treatment of cytochalasin D, which effectively inhibited invasion of L . pneumophila into A549, significantly reduced the production of IL-6 and TNF-alpha, but not IL-8 . These results suggested that the induction and expression of IL-6 and TNF-alpha in the human alveolar epithelial cells especially required intracellular signaling by L . pneumophila after invasion. Anesthesiology, 2005 Jan, 102(1), 63 - 68 Electronic Nose Prediction of a Clinical Pneumonia Score: Biosensors and Microbes; Hanson CW 3rd et al.; BACKGROUND:: The authors performed a prospective study to determine whether breath test analysis using an electronic nose correlates with a clinical pneumonia score . METHODS:: Exhaled gas was sampled from the expiratory limb of the ventilator in mechanically ventilated surgical intensive care patients and assayed with the electronic nose . Components of a clinical pneumonia score were recorded concurrently . RESULTS:: The score predicted by the electronic nose showed good correlation with the actual pneumonia score (r = 0.81) . Bland Altman analysis showed a mean bias of 0.0 (limits +/- 2.6) . CONCLUSIONS:: The electronic nose is a new biosensor technology that correlates with a clinical pneumonia score. EMBO J . 2004 Dec 23; {Epub ahead of print} The ancient origin of the complement system; Zhu Y et al.; The complement system has been thought to originate exclusively in the deuterostomes . Here, we show that the central complement components already existed in the primitive protostome lineage . A functional homolog of vertebrate complement 3, CrC3, has been isolated from a 'living fossil', the horseshoe crab (Carcinoscorpius rotundicauda) . CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes . CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system . Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor-like sequence . Furthermore, complement-mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed . Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species . Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals. Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 99 - 104 Epub 2004 Dec 22. Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury; Pull SL et al.; We have identified cellular and molecular features of the stem cell niche required for marked amplification of mouse colonic epithelial progenitors (ColEPs) that occurs in response to wounding of the epithelium with dextran sodium sulfate . This regenerative response in areas adjacent to breaches in the epithelial barrier depends on the gut microbiota because ColEP proliferation is markedly diminished in germ-free animals . Analysis of conventionally raised C57BL/6 (B6) knockout mice lacking the Toll-like receptor signal transduction pathway component Myd88 and wild-type animals transplanted with Myd88(-/-) bone marrow, revealed that Myd88-mediated signaling through mesenchymal cells is also required for the ColEP response . Studies of B6 Csf1(op/op) (lacking macrophages) mice, Rag1(-/-) mice, and wild-type mice treated with neutrophil-specific Gr1 mAbs, disclosed that macrophages but not lymphocytes or neutrophils are necessary . GeneChip analysis of laser-capture-microdissected mesenchymal cells coupled with immunohistochemical and electron microscopic studies showed that, during the regenerative response, macrophages in the pericryptal stem cell niche express genes associated with their activation and extend processes to directly contact ColEPs near the crypt base . GeneChip analysis also identified a number of potential molecular mediators of regeneration expressed in the pericryptal progenitor niche, including secreted factors that stimulate epithelial proliferation and proteins involved in extracellular matrix and basement membrane function, stability, and growth factor binding . Together, these studies indicate that the colonic epithelial progenitor niche is a dynamic structure in which macrophages function as mobile "cellular transceivers" that coordinate inputs from luminal microbes and injured epithelium and transmit regenerative signals to neighboring ColEPs. Appl Microbiol Biotechnol . 2004 Dec 22; {Epub ahead of print} Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12; Xiao-Lan L et al.; A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography . The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6% . The apparent molecular weight of the enzyme was 18.0 kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 16.6 kDa by gel filtration chromatography, which revealed a monomeric form of the enzyme . The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5+/-0.1 . The enzyme hydrolyzed fibrin . It cleaved the alpha, beta, and gamma chains of fibrinogen simultaneously, and it also hydrolyzed casein and N-succinyl-Ala-Ala-Pro-Phe-pNA . The enzyme had an optimal temperature of 45 degrees C, and an optimal pH of 10.5 . EDTA, PCMB, and PMSF inhibited the activity of the enzyme, and SBTI, Lys, TPCK, and Aprotinine had no obvious inhibition, which suggested that the activity center of the enzyme had hydrosulfuryl and metal . The first 12 amino acids of the N-terminal sequence of the enzyme were S-V-S-E-I-Q-L-M-H-N-L-G and had no homology with that of other fibrinolytic enzyme from other microbes. Shock, 2005 Jan, 23(1), 39 - 44 NEUTROPHIL ELASTASE, MIP-2, AND TLR-4 EXPRESSION DURING HUMAN AND EXPERIMENTAL SEPSIS; Tsujimoto H et al.; Highly activated neutrophils play a critical role in mediating organ injury in sepsis by releasing neutrophil elastase (NE) . Toll-like receptors (TLRs) play an important role in the host defense against invading microbes, and their signaling pathway is critical to the activation of the proinflammatory response . However, the relationship between TLR expression and the host defense mechanism during sepsis has not been fully elucidated . In this paper, we investigated the relationships among chemokine (MIP-2), TLR-4, and NE expression in human sepsis and murine peritonitis (CLP) . TLR-4 expression on monocytes/macrophages was examined in patients with sepsis and in murine peritonitis and was markedly increased in both populations . LPS-induced MIP-2 production by bronchoalveolar cells and liver mononuclear cells in mice with peritonitis was also significantly increased compared with sham-operated mice . Pretreatment of the macrophage cell line, RAW 264.7 cells, with a NE inhibitor before their exposure to LPS resulted in a significant dose-dependent decrease in MIP-2 production, which was comparable to that seen following pretreatment with TLR-4 antibody . Furthermore, NE and LPS both up-regulated TLR-4 expression on human peripheral blood monocytes . Thus, chemokine-induced recruitment of neutrophils in sepsis may result in further increased chemokine production and increased expression of TLR-4 . Neutrophil-derived NE may be associated with increased expression of monocyte/macrophage TLR-4, thereby serving as a positive feedback loop for the inflammatory response among the different cell populations. J Agric Food Chem, 2004 Dec 29, 52(26), 7822 - 7 Evaluation of metal and microbial contamination in botanical supplements; Raman P et al.; The sale of botanical dietary supplements in the United States is on the rise . However, limited studies have been conducted on the safety of these supplements . There are reports on the presence of undesired metals in some of the botanical dietary supplements . In this study, echinacea, garlic, ginkgo, ginseng, grape seed extract, kava kava, saw palmetto, and St . John's wort supplements manufactured by Nature's Way, Meijer, GNC, Nutrilite, Solaray, Sundown and Natrol, have been analyzed for lead, mercury, cadmium, arsenic, uranium, chromium, vanadium, copper, zinc, molybdenum, palladium, tin, antimony, thallium, and tungsten using inductively coupled plasma mass spectrometry . All samples were devoid of mercury contamination . Results indicated that the botanical supplements analyzed did not contain unacceptable concentrations of these metals . These supplements were also evaluated for microbial contamination, and most samples analyzed showed the presence of bacteria or fungi or both . Microbes were not counted nor were microbial counts determined in these samples. Acc Chem Res, 2004 Nov, 37(11), 902 - 8 Microbial detoxification of superoxide: the non-heme iron reductive paradigm for combating oxidative stress; Kurtz DM Jr; A reductive paradigm has emerged in recent years for detoxification of superoxide and other redox active diatomic molecules in air-sensitive bacteria and archaea . Adventitiously generated superoxide in many anaerobic or microaerophilic bacteria and archaea is scavenged by superoxide reductase (SOR) rather than the classical superoxide dismutases characteristic of aerobic microbes . SORs contain a novel five-coordinate, square-pyramidal {Fe(His)4(Cys)} ferrous active site, which adds a sixth glutamate ligand upon oxidation . This Account summarizes the recently elucidated structural and mechanistic features of SORs . The non-heme iron reductive scavenging paradigm in these air-sensitive microbes also extends to recently characterized enzymes that scavenge hydrogen peroxide and nitric oxide and to oxygen sensing proteins. Zh Obshch Biol, 2004 Nov-Dec, 65(6), 464 - 79 {Initiation of self-organization processes in community of marine microbenthos by application of nutrients in super-high concentrations}; Natural endogenous adjuvants; Department of Pathology, UMass Medical School, Worcester, MA, 01655, USA, Kenneth.rock@umassmed.eduIt has long been known that immunization with a protein by itself is often not sufficient to stimulate immunity, and may instead induce tolerance . To elicit productive immune responses exogenous adjuvants need to be co-injected with an antigen . One important class of adjuvants are the unique (non-mammalian) components of microbes . It is now believed that an adjuvant is required for immunity because the immune system evolved to respond to dangerous situations such as infections, and the presence of an adjuvant is the mechanism used to identify these situations . However, there are some circumstances where immune responses are generated in the apparent absence of any microbial or other exogenous adjuvant . Such situations include immune responses to transplants, tumors, autoimmunity and possibly certain viral infections . It has been postulated that in these situations the danger signals come from endogenous adjuvants that are released from dying cells . There is abundant evidence that dead cells are immunogenic, and recently it has been shown that cells contain endogenous adjuvant activities that are released after death . Some actual and putative endogenous adjuvants, such as monosodium urate and heat shock proteins, have been identified and there are others whose identities are not yet known . The potential biological roles of this class of adjuvants are discussed. Nat Mater . 2004 Dec 19; {Epub ahead of print} Self-assembly of polymeric microspheres of complex internal structures; Fialkowski M et al.; Self-assembly can easily produce intricate structures that would be difficult to make by conventional fabrication means . Here, self-assembly is used to prepare multicomponent polymeric microspheres of arbitrary internal symmetries . Droplets of liquid prepolymers are printed onto a water-soluble hydrogel, and are allowed to spread and coalesce into composite patches . These patches are then immersed in an isodense liquid, which both compensates the force of gravity and dissolves the gel beneath the polymers . Subsequently, the patches fold into spheres whose internal structures are dictated by the arrangement of the droplets printed onto the surface . The spheres can be solidified either thermally or by ultraviolet radiation . We present a theoretical analysis of droplet spreading, coalescence and folding . Conditions for the stability of the folded microspheres are derived from linear stability analysis . The composite microbeads that we describe are likely to find uses in optics, colloidal self-assembly and controlled-delivery applications. Nat Mater . 2004 Dec 19; {Epub ahead of print} Anisotropic particle synthesis in dielectrophoretically controlled microdroplet reactors; Millman JR et al.; The miniaturization of chemical and biological processes in microfluidic devices and bioarrays is a major technological achievement . Microchips performing multiphase material synthesis operations could be a future step in this trend of miniaturizing technology . Here we show how electrically controlled chips can be used for the synthesis and manipulation of new types of particles with advanced structure . The method is based on a technique that allows freely suspended droplets and particles to be entrapped and transported using electric fields . The fields that hold and guide the droplets and particles are applied through arrays of electrodes submerged in the oil . Each of the microdroplets suspended on the surface of fluorinated liquid serves as a microscopic reactor, where the particles are formed by solidification of the carrier droplets . Controlled on-chip assembly, drying, encapsulation and polymerization were used to make anisotropic 'eyeball' and striped particles, polymer capsules and semiconducting microbeads. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D329 - 33 EchoBASE: an integrated post-genomic database for Escherichia coli; Misra RV et al.; EchoBASE is a relational database designed to contain and manipulate information from post-genomic experiments using the model bacterium Escherichia coli K-12 . Its aim is to collate information from a wide range of sources to provide clues to the functions of the approximately 1500 gene products that have no confirmed cellular function . The database is built on an enhanced annotation of the updated genome sequence of strain MG1655 and the association of experimental data with the E.coli genes and their products . Experiments that can be held within EchoBASE include proteomics studies, microarray data, protein-protein interaction data, structural data and bioinformatics studies . EchoBASE also contains annotated information on 'orphan' enzyme activities from this microbe to aid characterization of the proteins that catalyse these elusive biochemical reactions. Mol Immunol, 2005 Feb, 42(4), 501 - 510 Activation of NK cell cytotoxicity; Smyth MJ et al.; Natural killer (NK) cells are innate effector lymphocytes necessary for defence against stressed, microbe-infected, or malignant cells . NK cells kill target cells by either of two major mechanisms that require direct contact between NK cells and target cells . In the first pathway, cytoplasmic granule toxins, predominantly a membrane-disrupting protein known as perforin, and a family of structurally related serine proteases (granzymes) with various substrate specificities, are secreted by exocytosis and together induce apoptosis of the target cell . The granule-exocytosis pathway potently activates cell-death mechanisms that operate through the activation of apoptotic cysteine proteases (caspases), but can also cause cell death in the absence of activated caspases . The second pathway involves the engagement of death receptors (e.g . Fas/CD95) on target cells by their cognate ligands (e.g . FasL) on NK cells, resulting in classical caspase-dependent apoptosis . The comparative role of these pathways in the pathophysiology of many diseases is being dissected by analyses of gene-targeted mice that lack these molecules, and humans who have genetic mutations affecting these pathways . We are also now learning that the effector function of NK cells is controlled by interactions involving specific NK cell receptors and their cognate ligands, either on target cells, or other cells of the immune system . This review will discuss the functional importance of NK cell cytotoxicity and the receptor/ligand interactions that control these processes. Biotechnol Lett, 2004 Nov, 26(22), 1707 - 11 Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by Streptomyces rimosus; Taylor AP et al.; A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth . Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures . Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture . Biomass solely within colloidal silica gels (1.7 mg ml(-1)), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml(-1)) yielded 27 and 21 mu g ml(-1) oxytetracycline compared with 97 and 104 mu g ml(-1) for unsterilised gels (3.6 and 5.2 mg ml(-1) biomass) displaying outgrowth . It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification. J Immunol Methods, 2004 Nov, 294(1-2), 53 - 66 A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation; Perruche S et al.; The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells) . Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice . This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples . A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss . Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis . We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry . The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery . This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice. Appl Microbiol Biotechnol, 2005 Jan, 66(4), 373 - 376 Epub 2004 Nov 16. HBMMD: an enhanced database of the microorganisms associated with deeper water marine invertebrates; Gunasekera AS et al.; The Harbor Branch Marine Microbial Database (HBMMD) provides preliminary taxonomic identifications and features of microorganisms maintained in the Harbor Branch Oceanographic Institution Marine Microbial Culture Collection . The microbes are primarily derived from marine invertebrates such as sponges (phylum Porifera) and soft corals (phylum Cnidaria) found in deep water environments {>120 feet (>35 m) seawater} . The microbes isolated from within marine invertebrates represent some unique taxa and phylogenetic signatures . The database provides a user-friendly method to systemically search or sort a desired input . The site allows a powerful search for multiple parameters of any entry . Images of the microbes are contained within the database and can be accessed from the website . The HBMMD homepage is located at http://www.hboi.edu/dbmr/dbmr_hbmmd.html. Chem Commun (Camb), 2004 Dec 21, (24), 2804 - 5 Epub 2004 Nov 18. Gold and silver nanoparticles functionalized with known numbers of oligonucleotides per particle for DNA detection; Chen Y et al.; The biospecificity of gold and silver nanoparticles, functionalized with known numbers of oligonucleotides, is demonstrated in colorimetric microbead assays for complementary and mismatch sequences. Crit Rev Biochem Mol Biol, 2004 May-Jun, 39(3), 165 - 95 Life with carbon monoxide; Ragsdale SW; This review focuses on how microbes live on CO as a sole source of carbon and energy and with CO by generating carbon monoxide as a metabolic intermediate . The use of CO is a property of organisms that use the Wood-L jungdahl pathway of autotrophic growth . The review discusses when CO metabolism originated, when and how it was discovered, and what properties of CO are ideal for microbial growth . How CO sensing by a heme-containing transcriptional regulatory protein activates the expression of CO metabolism-linked genes is described . Two metalloenzymes are the cornerstones of growth with CO: CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS) . CODH oxidizes CO to CO2, providing low-potential electrons for the cell, or alternatively reduces CO2 to CO . The latter reaction, when coupled to ACS, forms a machine for generating acetyl-CoA from CO2 for cell carbon synthesis . The recently solved crystal structures of CODH and ACS along with spectroscopic measurements and computational studies provide insights into novel bio-organometallic catalytic mechanisms and into the nature of a 140 A gas channel that coordinates the generation and utilization of CO . The enzymes that are coupled to CODH/ACS are also described, with a focus on a corrinoid protein, a methyltransferase, and pyruvate ferredoxin oxidoreductase. Alcohol, 2004 Jul, 33(3), 235 - 9 Acute exposure to ethanol affects Toll-like receptor signaling and subsequent responses: an overview of recent studies; Pruett SB et al.; Ethanol suppresses innate resistance to a variety of microbes, and findings of studies from both our laboratory and other laboratories indicate suppression of responses is mediated through two Toll-like receptors (TLRs): TLR3 and TLR4 . In this article, we review recent findings from studies in our laboratory, indicating that ethanol also suppresses responses mediated through other TLRs . Considering the importance of TLR-mediated responses in innate immunity, this supports the possibility that suppression of these responses may constitute a major mechanism by which ethanol suppresses innate immunity . In addition, ethanol-induced changes in cellular signaling and in patterns of gene expression induced through TLR3 were examined in mouse peritoneal macrophages, and these results are reviewed in this article . Signaling through TLR3 was inhibited, and results of DNA microarray analysis supported the notion that inhibition of an interferon-related amplification loop might be responsible for suppression of gene expression for several effector molecules of innate immunity and inflammation not previously known to be altered by ethanol . Thus, ethanol alters responses through most or all mouse TLRs, and this suppresses expression of a wide range of innate immune mediators. Arch Anim Nutr, 2004 Oct, 58(5), 343 - 52 Ruminal degradability of 15N labelled ribonucleic acid in grass; Schonhusen U et al.; The ruminal degradation of RNA in rye grass (Lolium perenne) was studied using the bag method . A non-lactating cow (BW 550 kg) fitted with a rumen cannula was used and fed twice daily at maintenance level with a chopped grass hay-based ration containing 30% ground barley . Rye grass, labelled during growth by fertilization with 15N2-urea (9.5 atom% 15N, 20 g N/m2), was cut at seven stages of growth and maturity and freeze-dried . RNA-N represented 6 to 17% of total N . Labelled grass samples (milled to 5.0 mm screen, 5.0+/-0.1 g DM) were incubated in polyester bags (100 x 200 mm, pore size 50 microm) in the rumen for periods of 1, 3, 6, 9, 12, 24, and 48 h . Data of N and RNA disappearances from the bags were fitted to an exponential equation to estimate parameters of degradation . The effective degradability of RNA in the rumen averaged 90+/-4%, for N it was 11% units lower (P < 0.001) . Degradability of RNA was correlated to that of N (R2 = 0.92) . Degradability of RNA (R2 = 0.96) and N (R2 = 0.93) decreased with increasing fibre content of grass . Increasing the fibre content by 1% diminished the degradability of RNA and N by 1.1% units and 2.4% units, respectively (P < 0.001) . Assuming a microbial protein synthesis in the rumen of 150 g/kg DOM, a N: RNA ratio of 1:1.35 in rumen microbes and a rumen outflow rate of 0.06 h(-1), a model calculation indicates that about 9 to 19% of duodenal RNA are of dietary origin in animals fed grass . This should be taken into account for the calculation of microbial N on the basis of RNA as marker. Biosens Bioelectron, 2005 Jan 15, 20(7), 1277 - 84 Scanning electrochemical microscopy for detection of biosensor and biochip surfaces with immobilized pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase as enzyme label; Zhao C et al.; Scanning electrochemical microscopy (SECM) was applied to study quinoprotein-based biosensor or biochip . A typical quinoprotein, pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH), was taken as example . Feedback mode and generation collection (GC) mode in SECM have been explored in imaging the catalytic activity of GDH on microscopic magnetic bead domains . Biotinylated GDH was immobilized by using streptavidin-coated paramagnetic microbeads, which were deposited as microspot on a hydrophobic surface . Ferrocenemethanol and ferricyanide were used as electron mediators for feedback and GC detection, respectively . Enzymatic catalysis was further studied quantitatively using the theory developed for SECM. Fungal Genet Biol, 2005 Jan, 42(1), 30 - 41 Truncated and RIP-degenerated copies of the LTR retrotransposon Pholy are clustered in a pericentromeric region of the Leptosphaeria maculans genome; Attard A et al.; The LMR1 5.2kb interspersed repeat of Leptosphaeria maculans was described by Taylor and Borgmann {Mol . Plant Microbe Interact . 7 (1994) 181} as an uncharacterized repeated element sharing homologies with both LINEs and SINEs . Here, we used the LMR1 sequence as a template to identify the full-length element within a 184-kb genomic sequence corresponding to the pericentromeric region of the 2.80Mb chromosome of isolate v23.1.3 . This region comprises (i) one 6980-bp full-sized Pholy element bordered by two 275- to 280-bp long terminal repeats (LTRs), (ii) five Pholy-related sequences, usually truncated at their 3' ends, and (iii) five solo-LTRs . Structural features strongly suggested that Pholy corresponds to an ancient copia-like retrotransposon, sharing strong homologies with the Elsa retrotransposon of Stagonospora nodorum . Pholy was also suggested to be specific to pericentromeric regions . Comparative analysis of the structure of the Pholy-like sequences occurring in the 184-kb contig and in other parts of the genome showed that this family of repeats is highly degenerated following extensive repeat induced point mutation (RIP). J Immunol, 2004 Dec 15, 173(12), 7444 - 53 Exploiting type 3 complement receptor for TNF-alpha suppression, immune evasion, and progressive pulmonary fungal infection; Brandhorst TT et al.; TNF-alpha is crucial in defense against intracellular microbes . Host immune cells use type 3 complement receptors (CR3) to regulate excess TNF-alpha production during physiological clearance of apoptotic cells . BAD1, a virulence factor of Blastomyces dermatitidis, is displayed on yeast and released during infection . BAD1 binds yeast to macrophages (Mphi) via CR3 and CD14 and suppresses TNF-alpha, which is required for resistance . We investigated whether blastomyces adhesin 1 (BAD1) exploits host receptors for immune deviation and pathogen survival . Soluble BAD1 rapidly entered Mphi, accumulated intracellularly by 10 min after introduction to cells, and trafficked to early and late endosomes . Inhibition of receptor recycling by monodansyl cadaverine blocked association of BAD1 with Mphi and reversed TNF-alpha suppression in vitro . Inhibition of BAD1 uptake with cytochalasin D and FcR-redirected delivery of soluble BAD1 as Ag-Ab complexes or of wild-type yeast opsonized with IgG similarly reversed TNF-alpha suppression . Hence, receptor-mediated entry of BAD1 is requisite in TNF-alpha suppression, and the route of entry is critical . Binding of soluble BAD1 to Mphi of CR3(-/-) and CD14(-/-) mice was reduced to 50 and 33%, respectively, of that in wild-type mice . Mphi of CR3(-/-) and CD14(-/-) mice resisted soluble BAD1 TNF-alpha suppression in vitro, but, in contrast to CR3(-/-) cells, CD14(-/-) cells were still subject to suppression mediated by surface BAD1 on wild-type yeast . CR3(-/-) mice resisted both infection and TNF-alpha suppression in vivo, in contrast to wild-type and CD14(-/-) mice . BAD1 of B . dermatitidis thus co-opts normal host cell physiology by exploiting CR3 to subdue TNF-alpha production and foster pathogen survival. Plant J, 2004 Dec, 40(6), 968 - 78 Mutations in PMR5 result in powdery mildew resistance and altered cell wall composition; Vogel JP et al.; Powdery mildews and other obligate biotrophic pathogens are highly adapted to their hosts and often show limited host ranges . One facet of such host specialization is likely to be penetration of the host cell wall, a major barrier to infection . A mutation in the pmr5 gene rendered Arabidopsis resistant to the powdery mildew species Erysiphe cichoracearum and Erysiphe orontii, but not to the unrelated pathogens Pseudomonas syringae or Peronospora parasitica . PMR5 belongs to a large family of plant-specific genes of unknown function . pmr5-mediated resistance did not require signaling through either the salicylic acid or jasmonic acid/ethylene defense pathways, suggesting resistance in this mutant may be due either to the loss of a susceptibility factor or to the activation of a novel form of defense . Based on Fourier transform infrared analysis, the pmr5 cell walls were enriched in pectin and exhibited a reduced degree of pectin modification relative to wild-type cell walls . In addition, the mutant had smaller cells, suggesting a defect in cell expansion . A double mutant with pmr6 (defective in a glycosylphosphatidylinositol-anchored pectate lyase-like gene) exhibited a strong increase in total uronic acid content and a more severe reduction in size, relative to the single mutants, suggesting that the two genes affect pectin composition, either directly or indirectly, via different mechanisms . These two mutants highlight the importance of the host cell wall in plant-microbe interactions. Int J Parasitol, 2004 Dec, 34(13-14), 1547 - 54 Variant genes and the spleen in Plasmodium vivax malaria; del Portillo HA et al.; It is generally accepted that Plasmodium vivax, the most widely distributed human malaria, does not cytoadhere in the deep capillaries of inner organs and thus this malaria parasite must have evolved splenic evasion mechanism in addition to sequestration . The spleen is a uniquely adapted lymphoid organ whose central function is the selective clearance of cell and other particles from the blood, and microbes including malaria . Splenomegaly is a hallmark of malaria and no other disease seems to exacerbate this organ as this disease does . Besides this major selective clearance function however, the spleen is also an erythropoietic organ which, under stress conditions, can be responsible for close to 40% of the RBC populations . Data obtained in experimental infections of human patients with P . vivax showed that anaemia is associated with acute and chronic infections and it has been postulated that the continued parasitemia might have been sufficient to infect and destroy most circulating reticulocytes . We review here the basis of our current knowledge of variant genes in P . vivax and the structure and function of the spleen during malaria . Based on this data, we propose that P . vivax specifically adhere to barrier cells in the human spleen allowing the parasite to escape spleen-clearance while favouring the release of merozoites in an environment where reticulocytes, the predominant, if not exclusive, host cell of P . vivax, are stored before their release into circulation to compensate for the anaemia associated with vivax malaria. Nat Med, 2004 Dec, 10(12), 1307 - 20 An innately interesting decade of research in immunology; Germain RN; "Nature has provided, in the white corpuscles as you call them-in the phagocytes as we call them-a natural means of devouring and destroying all disease germs . There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes." So opined B.B . in G.B . Shaw's The Doctor's Dilemma in a dramatic restatement of a key portion of Ilya Metchnikoff's Nobel Prize address: "Whenever the organism enjoys immunity, the introduction of infectious microbes is followed by the accumulation of mobile cells, of white corpuscles of the blood in particular which absorb the microbes and destroy them . The white corpuscles and the other cells capable of doing this have been designated 'phagocytes,' (i.e., devouring cells) and the whole function that ensures immunity has been given the name of 'phagocytosis'" . Based on these insights into the foundation of resistance to infectious disease, Metchnikoff was awarded the 1908 Nobel Prize in Physiology or Medicine together with Paul Ehrlich (Fig . 1) . Although both were cited for discoveries in immunity, the contributions of the two men seem worlds apart . Ehrlich's studies did not deal with generic responses to infection, but rather with the highly specific nature of antibodies and their relationship to the cells producing them: "As the cell receptor is obviously preformed, and the artificially produced antitoxin only the consequence, i.e . secondary, one can hardly fail to assume that the antitoxin is nothing else but discharged components of the cell, namely receptors discharged in excess" . But biological systems are just that-systems-and the parts need to work together . And so we arrive, a century later, at an appreciation for just how intimately related these two seemingly disparate aspects of host defense really are. Genetics, 2004 Nov, 168(3), 1443 - 55 Evolutionarily stable infection by a male-killing endosymbiont in Drosophila innubila: molecular evidence from the host and parasite genomes; Dyer KA et al.; Maternally inherited microbes that spread via male-killing are common pathogens of insects, yet very little is known about the evolutionary duration of these associations . The few examples to date indicate very recent, and thus potentially transient, infections . A male-killing strain of Wolbachia has recently been discovered in natural populations of Drosophila innubila . The population-level effects of this infection are significant: approximately 35% of females are infected, infected females produce very strongly female-biased sex ratios, and the resulting population-level sex ratio is significantly female biased . Using data on infection prevalence and Wolbachia transmission rates, infected cytoplasmic lineages are estimated to experience a approximately 5% selective advantage relative to uninfected lineages . The evolutionary history of this infection was explored by surveying patterns of polymorphism in both the host and parasite genomes, comparing the Wolbachia wsp gene and the host mtDNA COI gene to five host nuclear genes . Molecular data suggest that this male-killing infection is evolutionarily old, a conclusion supported with a simple model of parasite and mtDNA transmission dynamics . Despite a large effective population size of the host species and strong selection to evolve resistance, the D . innubila-Wolbachia association is likely at a stable equilibrium that is maintained by imperfect maternal transmission of the bacteria rather than partial resistance in the host species. Immunogenetics, 2004 Dec, 56(9), 667 - 74 Epub 2004 Dec. Genetic analysis of Toll/Interleukin-1 Receptor (TIR) domain sequences from rhesus macaque Toll-like receptors (TLRs) 1-10 reveals high homology to human TLR/TIR sequences; Sanghavi SK et al.; Toll-like receptors (TLRs) form a major group of pattern recognition receptors of the innate immune system that sense molecular patterns on microbes . The cytoplasmic Toll/Interleukin-1 Receptor (TIR) signaling domain is instrumental in inducing a signaling cascade upon recognition of specific ligands by TLRs . Because nonhuman primates are used as models of infectious and immune processes, we sought to obtain an increased understanding of nonhuman primate TLRs . We obtained the nucleotide sequences of the TIR domains of rhesus macaque TLRs 1-10 and examined their genetic relationships to TLRs from humans and mice . Alignment of the deduced amino acid sequences revealed macaque-specific changes mostly outside the conserved "Box" regions of the TLR/TIR domain . Assessment of mutational biases among TLRs from multiple species revealed a strong overall bias towards synonymous substitutions, with a few short regions showing evidence for positive selection outside the Box regions . This first presentation of the TLR/TIR domain sequences from nonhuman primates indicates that although there are species-specific differences, a high level of sequence homology exists in the critical signaling Box regions of macaque, human, and murine TLR/TIR domains . These findings suggest that animal models, including nonhuman primates, will be useful in modeling human TLR pathophysiology and therapy. Nat Med, 2004 Dec, 10(12 Suppl), S70 - 6 Social and environmental risk factors in the emergence of infectious diseases; Weiss RA et al.; Fifty years ago, the age-old scourge of infectious disease was receding in the developed world in response to improved public health measures, while the advent of antibiotics, better vaccines, insecticides and improved surveillance held the promise of eradicating residual problems . By the late twentieth century, however, an increase in the emergence and re-emergence of infectious diseases was evident in many parts of the world . This upturn looms as the fourth major transition in human-microbe relationships since the advent of agriculture around 10,000 years ago . About 30 new diseases have been identified, including Legionnaires' disease, human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), hepatitis C, bovine spongiform encephalopathy (BSE)/variant Creutzfeldt-Jakob disease (vCJD), Nipah virus, several viral hemorrhagic fevers and, most recently, severe acute respiratory syndrome (SARS) and avian influenza . The emergence of these diseases, and resurgence of old ones like tuberculosis and cholera, reflects various changes in human ecology: rural-to-urban migration resulting in high-density peri-urban slums; increasing long-distance mobility and trade; the social disruption of war and conflict; changes in personal behavior; and, increasingly, human-induced global changes, including widespread forest clearance and climate change . Political ignorance, denial and obduracy (as with HIV/AIDS) further compound the risks . The use and misuse of medical technology also pose risks, such as drug-resistant microbes and contaminated equipment or biological medicines . A better understanding of the evolving social dynamics of emerging infectious diseases ought to help us to anticipate and hopefully ameliorate current and future risks. J Allergy Clin Immunol, 2004 Dec, 114(6), 1441 - 8 Susceptibility to allergic lung disease regulated by recall responses of dual-receptor memory T cells; Aronica MA et al.; BACKGROUND: Microbial infections are associated with the initial susceptibility to and flares of asthma . However, immunologic mechanisms whereby infections might alter the asthmatic phenotype are lacking . OBJECTIVE: To test the hypothesis that memory T cells specific both for a viral antigen and an allergen could influence the pathogenesis of allergic disease in vivo . METHODS: We developed a system in which 2 distinct T-cell receptors coexist on the T-cell surface, 1 specific for a virus and the other for an inhaled antigen . RESULTS: We show that a population of dual-receptor T cells, polarized through a virus-specific T-cell receptor to contain T(H)1 or T(H)2 cells, can be reactivated through an unrelated T-cell receptor in recall responses in vivo . Quiescent memory cells derived from a T(H)1-polarized effector population blocked the development of airway hyperreactivity in a model of allergic lung disease, in association with decreased induction of chemokines and eosinophil recruitment . Conversely, reactivation of quiescent T(H)2 cells after inhalation of antigen or virus infection was sufficient to lead to the development of airway hyperresponsiveness and allergic pulmonary inflammation in mice whose lungs were previously normal . CONCLUSION: These data provide evidence that dual-receptor memory T cells can regulate allergic disease susceptibility and suggest that they may play a role in mediating the influence of microbes on asthma pathogenesis. Ambio, 2004 Nov, Spec No 13(13), 11 - 7 Mountain biodiversity, its causes and function; Korner C; The personal safety and well-being of one fifth, and water supply for almost half of all people depend directly or indirectly on the functional integrity of mountain ecosystems, the key component of which is a robust vegetation cover . The green 'coat' of the world's mountains is composed of specialized plants, animals and microbes, all nested in a great variety of microhabitats . Because a single mountain may host a series of climatically different life zones over short elevational distances, mountains are hot spots of biodiversity and priority regions for conservation . With their diverse root systems, plants anchor soils on slopes and prevent erosion . Both landuse and atmospheric changes such as elevated CO2 and climatic warming affect mountain biodiversity . Sustained catchment value depends on sustained soil integrity, which in turn depends on a diverse plant cover . Whether landuse in mountains is sustainable is a question of its consequences for water yield and biodiversity . Given their dependence on mountains, lowlanders should show concern for the highlands beyond their recreational value. Appl Environ Microbiol, 2004 Dec, 70(12), 6963 - 7 Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring; Agranovski IE et al.; A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field . The operational principle of the device is based on the passage of air through porous medium immersed in liquid . This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal . As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period . The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses . It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation . Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses . It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate . A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives . The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water . The same trend was observed throughout the entire 4-h experiment . There was no significant difference observed only for the robust vaccinia virus. Crit Rev Oral Biol Med, 2004, 15(6), 348 - 81 Pathogenesis of apical periodontitis and the causes of endodontic failures; Nair PN; Apical periodontitis is a sequel to endodontic infection and manifests itself as the host defense response to microbial challenge emanating from the root canal system . It is viewed as a dynamic encounter between microbial factors and host defenses at the interface between infected radicular pulp and periodontal ligament that results in local inflammation, resorption of hard tissues, destruction of other periapical tissues, and eventual formation of various histopathological categories of apical periodontitis, commonly referred to as periapical lesions . The treatment of apical periodontitis, as a disease of root canal infection, consists of eradicating microbes or substantially reducing the microbial load from the root canal and preventing re-infection by orthograde root filling . The treatment has a remarkably high degree of success . Nevertheless, endodontic treatment can fail . Most failures occur when treatment procedures, mostly of a technical nature, have not reached a satisfactory standard for the control and elimination of infection . Even when the highest standards and the most careful procedures are followed, failures still occur . This is because there are root canal regions that cannot be cleaned and obturated with existing equipments, materials, and techniques, and thus, infection can persist . In very rare cases, there are also factors located within the inflamed periapical tissue that can interfere with post-treatment healing of the lesion . The data on the biological causes of endodontic failures are recent and scattered in various journals . This communication is meant to provide a comprehensive overview of the etio-pathogenesis of apical periodontitis and the causes of failed endodontic treatments that can be visualized in radiographs as asymptomatic post-treatment periapical radiolucencies. Lab Chip, 2004 Dec, 4(6), 570 - 5 Epub 2004 Dec. Microchip-based enzyme-linked immunosorbent assay (microELISA) system with thermal lens detection; Sato K et al.; A microchip-based enzyme-linked immunosorbent assay (microELISA) system was developed and interferon-{gamma} was successfully determined . The system was composed of a microchip with a Y-shaped microchannel and a dam structure, polystyrene microbeads, and a thermal lens microscope (TLM) . All reactions required for the immunoassay were done in the microchannel by successive introduction of a sample and regents . The enzyme reaction product, in a liquid phase, was detected downstream in the channel using the TLM as substrate solution was injected . The antigen-antibody reaction time was shortened by the microchip integration . The limit of the determination was improved by adopting the enzyme label . Moreover, detection procedures were greatly simplified and required time for the detection was significantly cut . The system has good potential to be developed as a small and automated high throughput analyzer. Front Biosci, 2005 Jan 1, 10, 582 - 7 Print 2005 Jan 1. Inhibition of toll like receptor immune responses by microbial pathogens; Alvarez JI; Toll Like Receptors (TLRs) are pathogen recognition receptors (PRRs) that respond to specific pathogen associated molecular patterns (PAMPs) during microbial invasion . After TLR stimulation a series of cellular responses initiate an inflammatory response and influence specific adaptive immunity that ultimately destroy the pathogen . But the immune response is not always able to control the infection . Pathogens have developed mechanisms to overcome and evade distinct arms of vertebrate immunity . Many of these strategies have been extensively described, but with the recent discovery of TLRs additional means to manipulate the innate immune response are currently being studied . Pathogens generally inhibit TLR mediated immunity by either blocking signals that stimulate further host defense mechanisms or by down-regulating their expression . These inhibitory mechanisms have been mainly elucidated in bacterial systems, whereas in other microorganisms they remain to be identified . Here the strategies that pathogenic microbes use to subvert TLR mediated immune responses are reviewed. Clin Diagn Virol, 1996 May, 5(2-3), 191 - 6 The year of Pasteur: from the concept of antibody and antigen by Bordet (1895) to the ELISA . What future for immunological diagnosis? Pillot J. Background: Chronological account of the increase of the sensitivity of immunological reactions and future possible improvements are presented . Results: During these 100 years, the sensitivity of immunological reactions has been increased by approximately 5 log by reference to the basic technique of quantitative immunoprecipitation . Objectives: Future progresses can be foreseen (i) in the signal of labeled reagents, with the development of time-resolved fluoro-immunoassays; (ii) in the presentation of viral antigens on solid phase, with a larger use of polystyrene microbeads; (iii) in the antigen used for antibody detection, by promoting the characterization of antibodies to conformational structures of viruses; (iv) for antibody reagent preparations, particularly by immunomodulation for the development of antibodies to weak epitopes or to presently non-immunogenic structures and; (v) in the discrimination of detected antibodies . Characterization of the discontinuous or continuous nature of the recognized epitopes and of the affinity of antibodies could permit to date the infection . Inhibition of a monoclonal antibody reacting with a conformational epitope, and identification of an idiotope, will be more selective than the usual characterization of a large polyclonal antibody activity . Finally, the use of antibodies specific of the sole SIgA, molecule is expected to carry new informations in serological diagnosis at the entry of numerous infectious agents . ELISA techniques will become tightly complementary to PCR, which leads to an early diagnosis when the focus of infection is easily accessible, but rapidly looses its diagnosis capacity in acute infections. Electrophoresis, 2004 Nov, 25(21-22), 3730 - 9 Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker; Cho S et al.; A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads . A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer . This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min . The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively . In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip . RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification . As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase . The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase. J Exp Zoolog A Comp Exp Biol . 2004 Aug 16;301A(9):756-766 {Epub ahead of print} Nucleotide and protein sequences of a proteinase inhibitor from the vitelline envelope of dace (Tribolodon hakonensis) eggs; Hosomi O et al.; Our experimental purpose is to probe the structure(s) of the chorionic proteinase inhibitor and its cDNA sequence(s) and to develop the application of safe medicines for protection of human and other animal bodies from pathogenic microbe attacks . In this study, chorionic proteinase inhibitor protein was isolated, sequenced and used to base the design of PCR primers, which were then used to amplify DNA using RT-PCR . A cDNA clone of the protein which inhibited the activities of serine proteinases and thermolysin was obtained on the basis of mRNA extracted from ovarian tissue of dace, Tribolodon hakonensis, and the deduced amino acid sequence was determined . Chorionic proteinase inhibitor (TribSPI) peptides of about 9.0 kDa (TribSPI) and 14 kDa (TribSPI-S) were purified from vitelline envelope extracts by thermolysin-immobilized affinity-chromatography . The cloned TribSPI cDNA was 1,806 bp in length, and the open reading flame (ORF) was 915 bp encoding a protein of 305 amino acid residues . The inhibitor protein had a molecular mass of 33,550 daltons and was composed of five similar domains . Each domain contained eight cysteine residues, and it's deduced amino acid sequence was only 33 approximately 34% identical to those of human and porcine antileukoproteinases (hALP and pALP, respectively) . A possible binding-site for serine proteinases, Arg-Ile, was contained in three domains . J . Exp . Zool . 301A:756-766, 2004 . (c) 2004 Wiley-Liss, Inc. Can J Gastroenterol, 2004 Nov, 18(11), 671 - 5 D(-)-lactic acid-producing probiotics, D(-)-lactic acidosis and infants; Mack DR; There is mounting evidence that ingestion of selected probiotics can modify disease morbidity for specific conditions affecting humans, and there is growing interest in the amelioration or prevention of disease with probiotics . Modulation in gene expression of the cellular elements of the intestinal mucosa and interbacterial interactions are leading theories as to the mechanism whereby probiotics can effect benefit for the host . Furthermore, gene-environmental interactions are considered to be important in the development of disease in those at genetic risk . With the intestinal tract harbouring large numbers of bacteria, alteration of the microbial environment with probiotic microbes is being considered as a controllable factor that may limit disease expression for those at genetic risk . This reasoning has led to interest in the administration of probiotics to infants . However, there are significant developmental changes occurring in many organ systems from the time of parturition and during the first months of life . Because there is little in the published scientific medical literature regarding the effects of long-term administration of probiotics to infants, potential problems must be considered; one such issue is that of administration of D(-)-lactate-producing probiotics . An appraisal of the current knowledge of this potential adverse effect is the subject of this communication. Curr Dir Autoimmun, 2005, 8, 124 - 39 Dendritic cells control B cell growth and differentiation; Jego G et al.; We propose a model where human dendritic cell (DC) subsets control, in a coordinated fashion, B cell growth and differentiation . Plasmacytoid DCs drive memory B cell differentiation into effector plasma cell via type I interferon and IL-6 . Type I interferon activates myeloid DCs that regulate B cell priming and acquisition of memory phenotype via IL-12, IL-6 and Blys/Baff . This model also integrates the role of antigen-specific T cells activated by myeloid DCs . Thus, protective humoral immunity results from a highly coordinated interplay of human DC subsets . This interplay may explain the spreading of immune response to deal with antigenic drift and to maintain an active immunity against mutating microbe . It may also provide an explanation for spreading of the autoimmune repertoire as autoimmunity develops. J Virol, 2004 Dec, 78(24), 14023 - 32 Clinical latency and reactivation of AIDS-related mycobacterial infections; Shen Y et al.; The immune mechanisms associated with the evolution from latent to clinically active mycobacterial coinfection in human immunodeficiency virus type 1 (HIV-1)-infected humans remain poorly understood . Previous work has demonstrated that macaques infected with simian immunodeficiency virus (SIVmac) can develop persistent Mycobacterium bovis BCG coinfection and a fatal SIV-related tuberculosis-like disease by 4 months after BCG inoculation . In the present study, SIVmac-infected monkeys that developed clinically quiescent mycobacterial infection after BCG inoculation were followed prospectively for the reactivation of the BCG and the development of SIV-related tuberculosis-like disease . The development of clinically latent BCG coinfection in these SIVmac-infected monkeys was characterized by a change from high to undetectable levels of bacterial organisms, with or without measurable BCG mRNA expression in lymph node cells . The reactivation of clinically latent BCG coinfection and development of SIV-related tuberculosis-like disease were then observed in these SIVmac-BCG-coinfected monkeys during a 21-month period of follow-up . The reactivation of SIV-related tuberculosis-like disease in these animals coincided with a severe depletion of CD4 T cells and a loss of BCG-specific T-cell responses . Interestingly, bacterial superantigen challenge of the SIVmac-BCG-coinfected monkeys resulted in an up-regulation of clinically latent BCG coinfection, suggesting that infection with superantigen-producing microbes may increase the susceptibility of individuals to the reactivation of AIDS-related mycobacterial coinfection . Thus, reactivation of latent mycobacterial infections in HIV-1-infected individuals may result from a loss of T-cell immunity or from a superimposed further compromise of the immune system. Mol Ther, 2004 Dec, 10(6), 1140 - 2 Recombinant adenovirus vectors activate the alternative complement pathway, leading to the binding of human complement protein C3 independent of anti-ad antibodies; Jiang H et al.; Recombinant adenoviruses are one of the most common gene transfer vectors utilized in human clinical trials, but it is also clear that systemic administration of this virus will be met by host innate and adaptive antiviral immune responses . One element of innate immunity is the complement system, a group of proteins that has evolved to rapidly recognize foreign microbes and viruses and to clear them from the circulatory system prior to their gaining entry to vulnerable host cells . Excessive complement activation can initiate or propagate a number of deleterious inflammatory responses, by release of potent cytokines and anaphylatoxins and/or by direct cellular toxicity . These reactions can progress rapidly and are factors important in serious complications, including the systemic inflammatory response syndrome and the adult respiratory distress syndrome. Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17033 - 8 Epub 2004 Dec 7. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins; Blixt O et al.; Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide . The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids . The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses. J Agric Food Chem, 2004 Dec 1, 52(24), 7370 - 6 Influence of two insecticides, chlorpyrifos and quinalphos, on arginine ammonification and mineralizable nitrogen in two tropical soil types; Menon P et al.; Effects of seed treatments with chlorpyrifos {5 g of active ingredient (ai) kg(-1) of seed} and quinalphos (6.25 g of ai kg(-1) of seed) and standing crop treatments with chlorpyrifos (800 g of ai ha(-1)) and quinalphos (1000 g of ai ha(-1)) on arginine deamination and mineralizable nitrogen were monitored, in the sandy loam and loamy sand soils of two tropical semiarid fields, for three consecutive crop seasons . The arginine ammonification activity of rhizospheric microbes was inhibited after seed treatment with chlorpyrifos and quinalphos and their principal metabolites, 3,5,6-trichloro-2-pyridinol (TCP) and 3,5,6-trichloro-2-methoxypyridine (TMP) and 2-hydroxyquinoxaline and quinoxaline-2-thiol, respectively . Quinalphos produced transient inhibitions, whereas chlorpyrifos and its metabolites (TCP and TMP) exerted a greater inhibition in both loamy sand and sandy loam soils . Arginine ammonification by nonrhizospheric microbes was stimulated by standing crop treatments with both pesticides . In the loamy sand soil, the parent compounds stimulated rhizospheric N-mineralization, whereas the metabolites were inhibitory . However, nonrhizospheric N-mineralization was inhibited by both chlorpyrifos and quinalphos and stimulated by their metabolites . A higher magnitude of inhibition of arginine deamination in the loamy sand than in the sandy loam soil could be due to greater bioavailability of the pesticides in the former, resulting from lesser sorption of the pesticides due to alkalinity of the soil and its low content of clay and organic carbon . Although both pesticides affected mineralizable nitrogen, seed treatment with quinalphos and standing crop treatment with quinalphos and chlorpyrifos produced the most significant effects . The recommended doses of the pesticides not only efficiently controlled whitegrubs, which increased pod yields, but also left no residues in harvested kernels . They also caused no long-term inhibition of ammonification, which could have been of significant concern during the short crop period in semiarid areas where nitrogen determines plant productivity. J Exp Zoolog A Comp Exp Biol, 2004 Sep 1, 301(9), 756 - 66 Nucleotide and protein sequences of a proteinase inhibitor from the vitelline envelope of dace (Tribolodon hakonensis) eggs; Hosomi O et al.; Our experimental purpose is to probe the structure(s) of the chorionic proteinase inhibitor and its cDNA sequence(s) and to develop the application of safe medicines for protection of human and other animal bodies from pathogenic microbe attacks . In this study, chorionic proteinase inhibitor protein was isolated, sequenced and used to base the design of PCR primers, which were then used to amplify DNA using RT-PCR . A cDNA clone of the protein which inhibited the activities of serine proteinases and thermolysin was obtained on the basis of mRNA extracted from ovarian tissue of dace, Tribolodon hakonensis, and the deduced amino acid sequence was determined . Chorionic proteinase inhibitor (TribSPI) peptides of about 9.0 kDa (TribSPI) and 14 kDa (TribSPI-S) were purified from vitelline envelope extracts by thermolysin-immobilized affinity-chromatography . The cloned TribSPI cDNA was 1806 bp in length, and the open reading flame (ORF) was 915 bp encoding a protein of 305 amino acid residues . The inhibitor protein had a molecular mass of 33,550 daltons and was composed of five similar domains . Each domain contained eight cysteine residues, and it's deduced amino acid sequence was only 33 approximately 34% identical to those of human and porcine antileukoproteinases (hALP and pALP, respectively) . A possible binding-site for serine proteinases, Arg-Ile, was contained in three domains. Environ Toxicol Chem, 2004 Nov, 23(11), 2665 - 72 Pollution-induced community tolerance of soil microbes in response to a zinc gradient; Davis MR et al.; The long-term accumulation of Zn in soils provides the microbial community time to adapt to this heavy metal . To assess the effects of long-term exposure to Zn on the metabolic diversity and tolerance to Zn of soil microbial community, the pollution-induced community tolerance (PICT) method, based on the Biolog microtitre plate system, was used . It especially is useful to study gradients of pollutants for detecting PICT . Such a Zn gradient was found by selecting soils at increasing distances from galvanized electricity pylons at two sites where Zn accumulation had occurred over a period of decades . Soil metabolic profiles showed a humpbacked response to increasing soil Zn concentrations, indicating that accumulation of Zn up to 2,000 mg/kg did not decrease the metabolic biodiversity in the culturable fraction of the microbial community . This fraction of the microbial community showed significant PICT, because the effective concentration that reduces the metabolic biodiversity by 50% (EC50), values for Zn added to the Biolog wells increased by up to two orders of magnitude with increasing soil-Zn concentration along the transects . Significant PICT was detectable at soil Zn concentrations above approximately 300 mg/kg . The EC50 values correlated more closely with soil total Zn than with soil pore water Zn . The results suggest that, although long-term exposure to Zn imposes stress on soil microbes, resulting in an increased tolerance, detectable PICT does not necessarily lead to a decrease in metabolic diversity. Infect Immun, 2004 Dec, 72(12), 7275 - 81 Antifungal immune reactivity in nasal polyposis; Pitzurra L et al.; As a fungal etiology has been proposed to underlie severe nasal polyposis, the present study was undertaken to assess local antifungal immune reactivity in nasal polyposis . For this purpose, microbial colonization, along with the pattern of T helper 1 (Th1)/Th2 cytokine production and Toll-like receptor (TLR) expression, was evaluated in patients with nasal symptoms and with and without polyposis and in healthy subjects . The results show that Th2 reactivity was a common finding for patients with nasal polyposis regardless of the presence of microbes . The production of interleukin-10 was elevated in patients with bacterial and, particularly, fungal colonization, while both TLR2 expression and TLR4 expression were locally impaired in microbe-colonized patients . Eosinophils and neutrophils, highly recruited in nasal polyposis, were found to exert potent antifungal effector activities toward conidia and hyphae of the fungus and to be positively regulated by TLR2 or TLR4 stimulation . Therefore, a local imbalance between activating and deactivating signals to effector cells may likely contribute to fungal pathogenicity and the expression of local immune reactivity in nasal polyposis. J Immunol, 2004 Dec 1, 173(11), 6592 - 602 Plasmacytoid dendritic cell recruitment by immobilized CXCR3 ligands; Kohrgruber N et al.; Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense against them . The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokine receptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines . In this study, we demonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates the features of haptotaxis and chemorepulsion . To mediate "haptorepulsion" of pDCs, CXCR3 requires the encounter of its cognate ligands immobilized, optimally by heparan sulfate, in a form of a negative gradient . This is the first report of the absolute requirement of chemokine immobilization and presentation for its in vitro promigratory activity . The paradigmatic example of pDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used by other cells in response to other chemokines. FEMS Microbiol Lett, 2004 Dec 1, 241(1), 67 - 72 Endophytic naphthopyrone metabolites are co-inhibitors of xanthine oxidase, SW1116 cell and some microbial growths; Song YC et al.; Fractionation of the extract of Aspergillus niger . IFB-E003, an endophyte in Cyndon dactylon, gave four known compounds naphtho-gamma-pyrones rubrofusarin B, fonsecinone A, asperpyrone B and aurasperone A, which were further investigated biologically . Rubrofusarin B was shown to be cytotoxic to the colon cancer cell line SW1116 (IC50: 4.5 microgml-1), and aurasperone A inhibitory on XO (xanthine oxidase) (IC50: 10.9 micromoll-1) . Moreover, the four naphtho-gamma-pyrones exhibited growth inhibitions against the five test microbes with MICs ranging in between 1.9 and 31.2 microgml(-1) . The present recognition of rubrofusarin B and aurasperone A as strong co-inhibitors on XO, colon cancer cell and some microbial pathogens is of significance for the imperative discovery of new relevant therapeutic agents. Cell Mol Biol (Noisy-le-grand) . 2004 Dec 31;Suppl.50:OL515-OL516 {Epub ahead of print} ASYMMETRY ALTERS IMMUNE-PARTNER RELATIONSHIP; Rewald E et al.; Ambiguity in the mutual manipulation between the immune system, more appropriately termed integrated defense system, on the one hand and microbes and incipient tumors on the other, is to be taken into account . Such ambiguity may also apply to autoimmune infighting . Here we consider the association of its increased incidence with modern ways of living . Indifference about fate implies that longstanding, evolutionary regulated, immune/tissue relationships are no impediment for asymmetric estrangement promoted by ongoing globalization. Mikrobiol Z, 2004 Sep-Oct, 66(5), 30 - 9 {Plasmid spectra and phagotypes of Escherichia coli strains isolated from patients with large intestine tumours}; The interactions between human dendritic cells and microbes; possible clinical applications of dendritic cells; Division of Virology, National Center for Epidemiology, Budapest, HungaryThe dendritic cells comprise several subsets that induce and regulate the immune responses against foreign and self-antigens, and that can therefore function as initiators of protective immunity and inducers of central or peripheral tolerance . The different subpopulations of dendritic cells interact with and also influence other cell populations of the immune system, such as T and B lymphocytes and natural killer cells . The factors that determine the given dendritic cell functions depend on the state of maturation and the local microenvironment . The interactions between dendritic cells and microorganisms are rather complex, but progress in the past few years has shed light on several aspects of these interactions . This review lays emphasis on the interactions between human dendritic cells, important components of the intima of arterial specimens at areas predisposed to atherosclerotic lesions, and Chlamydia pneumoniae and cytomegalovirus, the human pathogens most strongly implicated in the development of atherosclerosis . In addition, several examples of the potential clinical applications of dendritic cells are described. J Appl Microbiol, 2004, 97(6), 1319 - 25 Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation; Elloway EA et al.; AIMS: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis . METHODS AND RESULTS: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins . Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry . Increasing concentrations of representative FITC-lectins, saturation binding and determination of K(d) and relative B(max) values were employed to characterize carbohydrate residue exposure . FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces . Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K(d) and relative B(max), which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence . CONCLUSIONS: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability . SIGNIFICANCE AND IMPACT OF THE STUDY: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner. Genetics . 2004 Nov 15; {Epub ahead of print} An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae; Arensburger P et al.; Transposable elements have proven invaluable tools for genetically manipulating a wide variety of plants, animals and microbes . Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae thereby disabling the mosquito's ability to transmit malaria . To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome . Endogenous, active class II transposable elements from A