Microorganisms

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%–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 Anopheles gambiae have not been exploited as gene vectors/drivers because none have been isolated . We report the discovery of an active class II transposable element, Herves, from the mosquito Anopheles gambiae . Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B . cucurbitae . Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D . melanogaster . Herves displays an altered target site preference from the distantly related hAT elements, Hermes and hobo . Herves is also present in An . arabiensis and An . merus with copy numbers similar to that found in An . gambiae . Preliminary data from an East African population is consistent with the element being transpositionally active in mosquitoes.

Insect Biochem Mol Biol, 2004 Dec, 34(12), 1297 - 304
A Drosophila salivary gland mucin is also expressed in immune tissues: evidence for a function in coagulation and the entrapment of bacteria; Korayem AM et al.; Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp150, an ecdysone-regulated mucin that is found in hemocytes, the gut (peritrophic membrane) and in the salivary glands . We are particularly interested in mucin immune functions and found that gp150 is released from larval hemocytes, becomes part of the clot and participates in the entrapment of bacteria . By RT-PCR and RNAi experiments, we identified gp150 as the previously described I71-7, an ecdysone-induced salivary glue protein . We discuss the evolutionary and biochemical implications of the dual use of salivary proteins for immune functions in insects . Further molecular characterization of such shared proteins may enable a better understanding of the properties of proteins involved in containment and elimination of microbes, as well as hemostasis and wound repair.

Trends Biotechnol, 2004 Dec, 22(12), 653 - 60
Impact of genomics on microbial food safety; Abee T et al.; Genome sequences are now available for many of the microbes that cause food-borne diseases . The information contained in pathogen genome sequences, together with the development of themed and whole-genome DNA microarrays and improved proteomics techniques, might provide tools for the rapid detection and identification of such organisms, for assessing their biological diversity and for understanding their ability to respond to stress . The genomic information also provides insight into the metabolic capacity and versatility of microbes; for example, specific metabolic pathways might contribute to the growth and survival of pathogens in a range of niches, such as food-processing environments and the human host . New concepts are emerging about how pathogens function, both within foods and in interactions with the host . The future should bring the first practical benefits of genome sequencing to the field of microbial food safety, including strategies and tools for the identification and control of emerging pathogens.

Int J Parasitol, 2004 Nov, 34(12), 1333 - 6
Amphidial neurons ADL and ASH initiate sodium dodecyl sulphate avoidance responses in the infective larva of the dog hookworm Anclyostoma caninum; Ketschek AR et al.; Ablations of specific amphidial neuron pairs with a laser microbeam were conducted to understand better the neurological basis of the behaviours of larval parasitic nematodes . To date, the functions of the amphidial neurons of Caenorhabditis elegans and their counterparts in parasitic nematodes have been found to be remarkably conserved allowing the possibility to predict the relationships between neurons and their functions . Therefore, we anticipated that ablation of neuron pairs ASH and ASK would abrogate avoidance of sodium dodecyl sulphate (SDS) by infective larvae (L3i) of Anclyostoma caninum . Instead, we have found that laser microbeam ablation of these neuron pairs did not eliminate SDS avoidance in A . caninum, but that neuron pairs ASH and ADL are the amphidial neurons responsible for SDS repulsion . When a droplet of the repellent is placed in the direct path of a normal A . caninum L3i, a strong backward avoidance response is triggered . However, when the ASH and ADL neurons are ablated, the nematodes demonstrate the opposite reaction, increasing their movement in a forward direction.

Scand J Immunol, 2004 Nov, 60(5), 463 - 70
High-polarity Mycobacterium avium-derived lipids interact with murine macrophage lipid rafts; Maldonado-Garcia G et al.; Cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are widely recognized as portals for pathogenic micro-organisms . A growing body of evidence demonstrates mobilization of host plasma cell membrane lipid rafts towards the site of contact with several pathogens as well as a strict dependence on cholesterol for appropriate internalization . The fate of lipid rafts once the pathogen has been internalized and the nature of the pathogen components that interact with them is however less understood . To address both these issues, infection of the J774 murine cell line with Mycobacterium avium was used as a model . After demonstrating that M . avium induces lipid raft mobilization and that M . avium infects J774 by a cholesterol-dependent mechanism, it is shown here that mycobacterial phagosomes harbour lipid rafts, which are, at least in part, of plasma cell membrane origin . On the other hand, by using latex microbeads coated with any of the three fractions of M . avium-derived lipids of different polarity, we provide evidence that high-polarity, in contrast to low-polarity and intermediate-polarity, mycobacterial lipids or uncoated latex beads have a strong capacity to induce lipid raft mobilization . These results suggest that high-polarity mycobacterial lipid(s) interact with host cell cholesterol-enriched microdomains which may in turn influence the course of infection.

Annu Rev Immunol . 2004 Nov 12; {Epub ahead of print}
Pentraxins at the Crossroads Between Innate Immunity, Inflammation, Matrix Deposition and Female Fertility; Garlanda C et al.; C reactive protein, the first innate immunity receptor identified, and serum amyloid P component are classic short pentraxins produced in the liver . Long pentraxins, including the prototype PTX3, are expressed in a variety of tissues . Some long pentraxins are expressed in the brain and some are involved in neuronal plasticity and degeneration . PTX3 is produced by a variety of cells and tissues, most notably dendritic cells and macrophages, in response to Toll-like receptor (TLR) engagement and inflammatory cytokines . PTX3 acts as a functional ancestor of antibodies, recognizing microbes, activating complement, and facilitating pathogen recognition by phagocytes, hence playing a nonredundant role in resistance against selected pathogens . In addition, PTX3 is essential in female fertility because it acts as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix . Thus, the prototypic long pentraxin PTX3 is a multifunctional soluble pattern recognition receptor at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility . Expected online publication date for the Annual Review of Immunology Volume 23 is March 19, 2005 . Please see for revised estimates.

Crit Rev Food Sci Nutr, 2004, 44(5), 379 - 407
Structural, biological, and evolutionary relationships of plant food allergens sensitizing via the gastrointestinal tract; Mills EN et al.; The recently completed genome sequence of the model plant species Arabidopsis has been estimated to encode over 25,000 proteins, which, on the basis of their function, can be classified into structural and metabolic (the vast majority of plant proteins), protective proteins, which defend a plant against invasion by pathogens or feeding by pests, and storage proteins, which proved a nutrient store to support germination in seeds . It is now clear that almost all plant food allergens are either protective or storage proteins . It is also becoming evident that those proteins that trigger the development of an allergic response through the gastrointestinal tract belong primarily to two large protein superfamilies: (1) The cereal prolamin superfamily, comprising three major groups of plant food allergens, the 2S albumins, lipid transfer proteins, and cereal alpha-amylase/trypsin inhibitors, which have related structures, and are stable to thermal processing and proteolysis . They include major allergens from Brazil nut, peanuts, fruits, such as peaches, and cereals, such as rice and wheat; (2) The cupin superfamily, comprising the major globulin storage proteins from a number of plant species . The globulins have been found to be allergens in plant foods, such as peanuts, soya bean, and walnut; (3) The cyteine protease C1 family, comprising the papain-like proteases from microbes, plants, and animals . This family contains two notable allergens that sensitize via the GI tract, namely actinidin from kiwi fruit and the soybean allergen, Gly m Bd 30k/P34 . This study describes the properties, structures, and evolutionary relationships of these protein families, the allergens that belong to them, and discusses them in relation to the role protein structure may play in determining protein allergenicity.

Clin Diagn Lab Immunol, 2004 Nov, 11(6), 1094 - 9
Recombinant Helicobacter bilis protein P167 for mouse serodiagnosis in a multiplex microbead assay; Feng S et al.; Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies . Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers . This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H . bilis infection . The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format . Individual microbead sets were conjugated to purified, H . bilis-specific, recombinant proteins P167C and P167D and bacterial membrane extracts from H . bilis and Helicobacter hepaticus . For detecting H . bilis infection in the microbead multiplex assay, P167C and P167D provided significantly higher sensitivities (94 and 100%, respectively) and specificities (100 and 95%, respectively) than membrane extract (78% sensitivity and 65% specificity) . Microbead multiplex assay results were validated by enzyme-linked immunosorbent assay . Purified recombinant proteins showed low batch-to-batch variation; this feature allows for ease of quality control, assay robustness, and affordability . Thus, recombinant antigens are highly suitable in the multiplex microbead assay format for serodetection of H . bilis infection.

Trends Microbiol, 2004 Dec, 12(12), 529 - 32
Battling enteroinvasive bacteria: Nod1 comes to the rescue; Chamaillard M et al.; Recognition of pathogenic bacteria by mammalian hosts is largely mediated by membrane-bound Toll-like receptors (TLRs) . Recently, a family of cytosolic proteins, termed NODs, with homology to plant disease-resistance gene products has been implicated in sensing microbes within the cytosol . The role of NOD family members in host defense is largely unknown . However, a recent report revealed that Nod1 is a crucial sensor for certain enteroinvasive bacteria that avoid TLR signaling . This finding suggests that Nod1 plays an important role in the initial recognition of pathogenic bacteria at epithelial surfaces, such as the gut, where innate immune responses to commensal bacteria must be avoided.

J Biol Rhythms, 2004 Oct, 19(5), 414 - 24
The neurospora circadian system; Dunlap JC et al.; The eukaryotic filamentous fungus Neurospora crassa has proven to be a durable and dependable model system for the analysis of the cellular and molecular bases of circadian rhythms . Pioneering genetic analyses identified clock genes, and beginning with the cloning of frequency (frq), work over the past 2 decades has revealed the molecular basis of a core circadian clock feedback loop that has illuminated our understanding of circadian oscillators in microbes, plants, and animals . In this transcription/translation-based feedback loop, a heterodimer of the White Collar-1 (WC-1) and WC-2 proteins acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene . FRQ dimerizes and feeds back to block the activity of its activators (making a negative feedback loop), as well as feeding forward to promote the synthesis of its activator, WC-1 . Phosphorylation of FRQ by several kinases leads to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle . Light resetting of the clock can be understood through the rapid light induction of frq expression and temperature resetting through the influence of elevated temperatures in driving higher levels of FRQ . Several FRQ- and WC-independent, noncircadian FRQ-less oscillators (FLOs) have been described, each of which appears to regulate aspects of Neurospora growth or development . Overall, the FRQ/white collar complex feedback loop appears to coordinate the circadian system through its activity to regulate downstream-target clock-controlled genes, either directly or via regulation of driven FLOs.

J Acoust Soc Am, 2004 Oct, 116(4 Pt 1), 2324 - 37
Secant lines in a preliminary study of phonetic reduction of /I/ and /epsilon/; McGowan RS; Two subjects from the X-Ray Microbeam Speech Production Database were examined in their production of the vowels /I/ and /epsilon/ in alveolar and dental consonant contexts . Secant lines, or first-order splines, between the three most anterior pellets were examined at vowel critical times . These critical times were zero crossings in the tangential acceleration of the midpoints of the secant lines . We expected and found, in general, that vowel reduction occurred as a function of vowel duration in measures of the secant line midpoint-to-palate distance and secant line orientation at vowel critical times . The shorter the vowel, the smaller the distance of the secant line midpoints to the palate and the less downward the orientation of the secant lines at the vowel critical times . Phonetic reduction was also apparent in the formant frequencies . There were differences between the speakers in terms of the range of vowel duration and degree of reduction . The subjects differed in the functional parts of the tongue spanned by the secant lines and the shape of their palates . These differences were factors in the observed relations between formant frequencies and the articulatory, secant line measures for each subject.

Biotechnol Bioeng, 2004 Dec 20, 88(6), 740 - 9
Towards a fully synthetic substitute of alginate: optimization of a thermal gelation/chemical cross-linking scheme ("tandem" gelation) for the production of beads and liquid-core capsules; Cellesi F et al.; Fully synthetic polymers were used for the preparation of hydrogel beads and capsules, in a processing scheme that, originally designed for calcium alginate, was adapted to a "tandem" process, that is the combination a physical gelation with a chemical cross-linking.The polymers feature a Tetronic backbone (tetra armed Pluronics), which exhibits a reverse thermal gelation in water solutions within a physiological range of temperatures and pHs . The polymers bear terminal reactive groups that allow for a mild, but effective chemical cross-linking . Given an appropriate temperature jump, the thermal gelation provides a hardening kinetics similar to that of alginate . With slower kinetics, the chemical cross-linking then develops an irreversible and elastic gel structure, and determines its transport properties . In the present article this process has been optimized for the production of monodisperse, high elastic, hydrogel microbeads, and liquid-core microcapsules . We also show the feasibility of the use of liquid-core microcapsules in cell encapsulation . In preliminary experiments, CHO cells have been successfully encapsulated preserving their viability during the process and after incubation . The advantages of this process are mainly in the use of synthetic polymers, which provide great flexibility in the molecular design . This, in principle, allows for a precise tailoring of mechanical and transport properties and of bioactivity of the hydrogels, and also for a precise control in material purification.

Trends Immunol, 2004 Dec, 25(12), 687 - 93
TLRs: Professor Mechnikov, sit on your hat; O'Neill LA; Toll-like receptors (TLRs) are sensors of foreign microbial products, which initiate host defense responses in all multicellular organisms examined to date . They are the target for most adjuvants, are essential for the establishment of memory in T and B cells and provoke inflammation . They program dendritic cells in their interaction with Th1 cells and their signalling pathways enable a tailoring of host defense responses to the provoking microbe previously unsuspected in the innate arm of immunity . Their discovery and characterisation fills a void in immunology and is the culmination of an effort that began with one of immunology's founding fathers, Elie Mechnikov . Targeting TLRs therapeutically now has the potential to impact on how we treat infectious and inflammatory diseases.

Appl Environ Microbiol, 2004 Nov, 70(11), 6670 - 7
Life history implications of rRNA gene copy number in Escherichia coli; Stevenson BS et al.; The role of the rRNA gene copy number as a central component of bacterial life histories was studied by using strains of Escherichia coli in which one or two of the seven rRNA operons (rrnA and/or rrnB) were deleted . The relative fitness of these strains was determined in competition experiments in both batch and chemostat cultures . In batch cultures, the decrease in relative fitness corresponded to the number of rRNA operons deleted, which could be accounted for completely by increased lag times and decreased growth rates . The magnitude of the deleterious effect varied with the environment in which fitness was measured: the negative consequences of rRNA operon deletions increased under culture conditions permitting more-rapid growth . The rRNA operon deletion strains were not more effective competitors under the regimen of constant, limited resources provided in chemostat cultures . Enhanced fitness in chemostat cultures would have suggested a simple tradeoff in which deletion strains grew faster (due to more efficient resource utilization) under resource limitation . The contributions of growth rate, lag time, Ks, and death rate to the fitness of each strain were verified through mathematical simulation of competition experiments . These data support the hypothesis that multiple rRNA operons are a component of bacterial life history and that they confer a selective advantage permitting microbes to respond quickly and grow rapidly in environments characterized by fluctuations in resource availability.

Blood Cells Mol Dis, 2004 Nov-Dec, 33(3), 281 - 7
Long-term outcome after haploidentical stem cell transplantation in children; Lang P et al.; We present an update of our results with transplantation of highly purified stem cells from one to three loci mismatched parental donors . Sixty-three pediatric patients with acute lymphoblastic leukemias (n = 32), acute myeloid, chronic myeloid and myelomonocytic leukemias (n = 13), myelodysplastic syndromes (n = 4), lymphomas (n = 4), and various nonmalignant diseases (n = 10) underwent transplantation . Mobilized peripheral-blood stem cells were selected with either anti-CD34- or anti-CD133-coated microbeads . Patients received a median of 19.5 x 10(6) purified cells and <25,000 CD3+ T lymphocytes per kilogram, with no regular posttransplant pharmacological immunosuppression . Engraftment occurred in 98% of patients (primary sustained engraftment, 83%; engraftment after reconditioning/stem cell boosts, 15%) . Moreover, all survivors but one had a stable three-lineage engraftment with a median follow up of 4.1 years (range 0.6-8 years) . Primary acute graft-versus-host disease (GvHD) grade II was seen in only 7% of patients . No severe primary acute GvHD grades III-IV occurred . Thirteen percent of the patients developed transient chronic GvHD . Probability of disease-free survival (DFS) at 3 years was 60% for patients with nonmalignant diseases and 48% for patients with acute lymphatic leukemia (ALL)/non-Hodgkin lymphoma (NHL) in complete remission (CR)1-3 . None of the ALL/NHL patients with active disease survived . Children with acute and chronic myeloid leukemias had a poorer outcome (3-year DFS = 18%), whereas two of four patients with myelodysplastic syndrome (MDS) are alive . Relapse probability of the whole group was not significantly increased when compared to a historical control group . The incidence of lethal viral infections was 18% between 1995 and 2002 and has since been reduced to 8% by the introduction of new therapeutic strategies . In summary, the use of stem cells from haploidentical parental donors should be strongly considered in all children who need transplantation but lack an identical donor.

Semin Immunol, 2004 Oct, 16(5), 323 - 33
Generation and maintenance of immunological memory; Gourley TS et al.; The key feature of the adaptive immune response is its specificity and the ability to generate and maintain memory . Preexisting antibodies in the circulation and at the mucosa provide the first line of defense against re-infection by extracellular as well as intracellular pathogens . Memory T cells are an important second line of defense against intracellular pathogens, and in particular against microbes that can cause chronic or latent infection . In this article we will review our current understanding of the generation and maintenance of B cell and T cell memory.

In Vivo, 2004 Sep-Oct, 18(5), 629 - 32
Use of annexin V for the identification of fetal cells in maternal circulation; Kolialexi A et al.; MACS with Annexin V-conjugated microbeads was used to isolate cells in apoptosis from the peripheral blood of 112 women at different weeks of gestation and from 15 women (60 samples) sequentially tested postpartum . FISH using X/Y probes was applied to quantitate fetal apoptotic cells . The mean apoptosis rate in the 16th-18th week was 6.5% and fetal cells constituted 5.1% of the apoptotic cell population . In the 26th-28th week it was 10.1%/7.5% and in the 37th-38th week 12.5%/9.9%, respectively . In samples obtained 30 min, 12h and 24h postpartum, the mean apoptosis was 25.1%, 12.5 and 6.1%, respectively and fetal cells constituted 14.8%, 2.1% and 0.16% of the apoptotic cells . Forty-eight h after delivery, apoptosis was 2.3% and no fetal cells were detected . Accurate estimation of the proportion of fetal cells undergoing apoptosis may facilitate the optimization of protocols for non-invasive prenatal diagnosis of chromosomal abnormalities.

Biosens Bioelectron, 2004 Nov 1, 20(4), 887 - 94
Fabrication of comb interdigitated electrodes array (IDA) for a microbead-based electrochemical assay system; Kim SK et al.; This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase . Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured . ANSYS was used to model steady state currents for electrodes with different geometries . Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation . Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs . The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase . We conclude that faster and more sensitive assays can be achieved with the comb IDA.

J Clin Invest, 2004 Nov, 114(9), 1222 - 6
Homeostatic control of immunity by TCR peptide-specific Tregs; Kumar V; Regulation of the immune response is a multifaceted process involving lymphocytes that function to maintain both self tolerance as well as homeostasis following productive immunity against microbes . There are 2 broad categories of Tregs that function in different immunological settings depending upon the context of antigen exposure and the nature of the inflammatory response . During massive inflammatory conditions such as microbial exposure in the gut or tissue transplantation, regulatory CD4+CD25+ Tregs broadly suppress priming and/or expansion of polyclonal autoreactive responses nonspecifically . In other immune settings where initially a limited repertoire of antigen-reactive T cells is activated and expanded, TCR-specific negative feedback mechanisms are able to achieve a fine homeostatic balance . Here I will describe experimental evidence for the existence of a Treg population specific for determinants that are derived from the TCR and are expressed by expanding myelin basic protein-reactive T cells mediating experimental autoimmune encephalomyelitis, an animal prototype for multiple sclerosis . These mechanisms ensure induction of effective but appropriately limited responses against foreign antigens while preventing autoreactivity from inflicting escalating damage . In contrast to CD25+ Tregs, which are most efficient at suppressing priming or activation, these specific Tregs are most efficient in controlling T cells following their activation.

Genome Res, 2004 Nov, 14(11), 2268 - 72
Properties of overlapping genes are conserved across microbial genomes; Johnson ZI et al.; There are numerous examples from the genomes of viruses, mitochondria, and chromosomes that adjacent genes can overlap, sharing at least one nucleotide . Overlaps have been hypothesized to be involved in genome size minimization and as a regulatory mechanism of gene expression . Here we show that overlapping genes are a consistent feature (approximately one-third of all genes) across all microbial genomes sequenced to date, have homologs in more microbes than do non-overlapping genes, and are therefore likely more conserved . In addition, the size, phase (reading frame offset), and distribution, among other characteristics, of overlapping genes are most consistent with the hypothesis that overlaps function in the regulation of gene expression . The upstream sequences and conservation of overlapping orthologs of two model organisms from the genus Prochlorococcus that have significantly different GC-content, and therefore different nucleotide sequences for orthologs, are also consistent with small overlapping sequence regions and programmed shifts in reading frame as a common mechanism in the regulation of microbial gene expression.

Lab Invest . 2004 Nov 01; {Epub ahead of print}
Oligoclonal T cells in histiocytic necrotizing lymphadenopathy are associated with TLR9(+) plasmacytoid dendritic cells; Lin CW et al.; Histiocytic necrotizing lymphadenopathy (HNL), a disease of unknown cause, is characterized pathologically by the presence of plasmacytoid dendritic cells (pDCs), which are frequently mixed with oligoclonal T cells (OTCs) and myeloid cells . Toll-like receptors (TLRs 1-10) are a family of pattern recognition receptors of DCs . To investigate the interactions between pDCs and T cells, and to look for an etiology of HNL, we studied 24 HNLs for the profile of TLRs . Transcripts of TLR7, a receptor on pDCs for single-stranded RNA, were found in every case, confirming the universal presence of pDCs . Transcripts of TLR9, another receptor on pDCs for microbial unmethylated CpG-rich DNA, were correlated with OTCs, implying T-cell expansion stimulated by TLR9(+) pDCs in response to a microbe . Because PCRs for bacterial 16S rDNAs were negative in the lymph nodes, a bacterial origin seems unlikely, but a virus remains a possible candidate . The pDCs lacked the maturation marker CD83, which suggested ineffective stimulation of T cells and might account for the usually benign course of HNL . Taken together, these data illustrate a novel approach, based upon TLR transcript analysis, for the integration of pathology, immunology, and clinical findings of HNL.Laboratory Investigation advance online publication, 1 November 2004; doi:10.1038/labinvest.3700201.

Anal Chem, 2004 Nov 1, 76(21), 6239 - 46
Manipulation of self-assembled structures of magnetic beads for microfluidic mixing and assaying; Rida A et al.; We present an original concept of manipulation of magnetic microbeads in a microchannel . It is based on the dynamic motion of a self-assembled structure of ferrimagnetic beads that are retained within a microfluidic flow using a local alternating magnetic field . The latter induces a rotational motion of the magnetic particles, thereby strongly enhancing the fluid perfusion through the magnetic structure that behaves as a dynamic random porous medium . The result is a very strong particle-liquid interaction that can be controlled by adjusting the magnetic field frequency and amplitude, as well as the liquid flow rate, and is at the basis of very efficient liquid mixing . The principle is demonstrated using a microfluidic chip made of poly(methyl methacrylate) with integrated soft ferromagnetic plate structures . The latter are part of an electromagnetic circuit and serve to locally apply a magnetic field over the section of the microchannel . Starting from a laminar flow pattern of parallel fluorescein dye and nonfluorescent liquid streams, we demonstrate a 95% mixing efficiency using a mixing length of only 400 microm and at liquid flows of the order of 0.5 cm/s . We anticipate that the intense interaction between the fluid and magnetic particles with functionalized surfaces holds large potential for the development of future bead-based assays.

Scand J Gastroenterol, 2004 Oct, 39(10), 981 - 7
Extensive homology between the major immunodominant mitochondrial antigen in primary biliary cirrhosis and Helicobacter pylori does not lead to immunological cross-reactivity; Bogdanos DP et al.; BACKGROUND: Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic disease characterized by the presence of antibodies directed predominantly against the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) . What provokes tolerance breakdown in PBC remains to be established, though there is evidence to indicate that microbes may induce anti-mitochondrial antibodies (AMA) through a mechanism of molecular mimicry . METHODS: Having found that urease beta (UREB)(22-36) antigen of Helicobacter pylori (HELPY) shares extensive (87%) similarity with PDC-E2(212-226), the major mitochondrial autoepitope, it was hypothesized that this would also lead to cross-reactivity . The UREB/PDC-E2 mimics were thus constructed and tested by ELISA in 112 PBC patients and 114 controls . RESULTS: Reactivity to PDC-E2(212-226) was found in 104 patients but to UREB(22-36) in only 2 . In these two patients, the double reactivity was not cross-reactive . The lack of surface antibody accessibility to UREB(22-36), as demonstrated through three-dimensional model prediction analysis, may explain this unexpected finding . There was some speculation on whether HELPY UREB(22-36) might act as a cross-reactive CD4 T-cell epitope . All seven PBC patients, tested in a standard proliferation assay against PDC-E2(212-226), gave a positive response . All seven were unresponsive to HELPY UREB(22-36) . The pattern of reactivity to HELPY antigens by immunoblot was similar between anti-PDC-E2-positive and negative PBC cases, as well as between PBC patients and controls . CONCLUSION: Contrary to common belief, extensive sequence homology (molecular mimicry) between self and microbe does not necessarily result in cross-reactivity . It is therefore likely that, when present, cross-reactivity between self and microbes is of biological importance.

Nihon Hansenbyo Gakkai Zasshi, 2004 Sep, 73(3), 263 - 70
{Molecular pathogenesis of mycobacterial diseases}; Kobayashi K; Mycobacterial diseases, including tuberculosis, leprosy, and disease due to nontuberculous mycobacteria, are the major cause of death from infectious diseases around the world . About one-third of the world population is latently infected with Mycobacterium tuberculosis . Over 8 million new cases and nearly 2 million deaths occur each year . Tuberculosis presents a significant health threat to the world . The pathogenicity of mycobacteria is related to their ability to escape killing by ingested macrophages, latent infection, and induce delayed type hypersensitivity . This has been attributed to several components of the mycobacterial cell wall, such as surface glycolipids, lipoarabinomannan, complement activation factor, heat-shock protein, and mycobacterial DNA binding protein . From the aspect of my research interests, I have focused on mycobacterial glycolipids and mycobacterial DNA binding protein in this article . Surface molecules of mycobacteria exert pleiotropic activities in both the microbe and host, and thus participate in the pathogenesis of mycobacterial diseases . The better understanding of mycobacterial pathogenicity may open the new avenue for the development of therapeutic and prophylactic interventions.

Med Sci Monit, 2004 Nov, 10(11), RA255 - 60 Epub 2004 Oct 26.
Atherosclerosis: immunopathogenesis and immunotherapy; Ohashi R et al.; Atherosclerosis is a chronic disease that causes various cardiovascular complications . Although the initiation and progression of atherosclerosis largely depend on genetic factors and life styles, the cellular and molecular mechanisms are still not clear . Recent studies have revealed that cellular and humoral immunity plays crucial roles in atherogenic lesion formation, including macrophages, CD4+ and CD8+ T cells and dendritic cells as well as autoantigens such as heat shock protein (HSP 60/65) and oxidized LDL . Furthermore, atherosclerosis is associated with microbial or viral infection . Given these recent advances, various modifications of the immune system in mouse models have been performed to determine the underlying mechanisms of atherogenesis and new therapeutic strategies . Blocking of macrophage inducing factors or disruption of scavenger receptors on macrophages such as SR-A and CD36 can inhibit atherosclerosis progression . Switching the immune system of CD4+ T cells from Th1 to Th2 can induce secretion of anti-inflammatory cytokine IL-10, leading to decreased atherosclerotic lesions . Eradication of microbes and viruses can also reduce atherosclerosis . These investigations strongly support that immune responses are important mechanisms of atherogenesis, and immunomodulation can be a new strategy to treat atherosclerosis.

J Endod, 2004 Nov, 30(11), 782 - 4
Bacterial leakage with mineral trioxide aggregate or a resin-modified glass ionomer used as a coronal barrier; Tselnik M et al.; The purpose of this study was to evaluate gray mineral trioxide aggregate (MTA), white MTA, and Fuji II LC cement as coronal barriers to bacterial leakage . Seventy-eight, matched, human teeth were obturated with gutta-percha . In group I, 18 pairs received a 3-mm barrier of gray or white MTA . In group II, 18 pairs received a 3-mm barrier of gray MTA or Fuji cement . Three pairs were used as positive (obturated without barrier) and negative (covered with epoxy resin) controls . A dual-chamber leakage model utilizing salivary microbes was used for the evaluation . Leakage was recorded when turbidity was observed . All controls behaved as expected . In group I, three gray MTA and three white MTA samples leaked . In group II, one gray MTA and three Fuji samples leaked . There was no statistically significant difference in leakage between gray and white MTA or gray MTA and Fuji at 30, 60, or 90 days . Gray and white MTA or Fuji II can be recommended as a coronal barrier for up to 3 months.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15609 - 14 Epub 2004 Oct 25.
EncM, a versatile enterocin biosynthetic enzyme involved in Favorskii oxidative rearrangement, aldol condensation, and heterocycle-forming reactions; Xiang L et al.; The bacteriostatic natural product enterocin from the marine microbe "Streptomyces maritimus" has an unprecedented carbon skeleton that is derived from an aromatic polyketide biosynthetic pathway . Its caged tricyclic, nonaromatic core is derived from a linear poly-beta-ketide precursor that formally undergoes a Favorskii-like oxidative rearrangement . In vivo characterization of the gene encM through mutagenesis and heterologous biosynthesis demonstrated that its protein product not only is solely responsible for the oxidative C-C rearrangement, but also facilitates two aldol condensations plus two heterocycle forming reactions . In total, at least five chiral centers and four rings are generated by this multifaceted flavoprotein . Heterologous expression of the enterocin biosynthesis genes encABCDLMN in Streptomyces lividans resulted in the formation of the rearranged metabolite desmethyl-5-deoxyenterocin and the shunt products wailupemycins D-G . Addition of the methyltransferase gene encK, which was previously proposed through mutagenesis to additionally assist EncM in the Favorskii rearrangement, shifted the production to the O-methyl derivative 5-deoxyenterocin . The O-methyltransferase EncK seems to be specific for the pyrone ring of enterocin, because bicyclic polyketides bearing pyrone rings are not methylated in vivo . Expression of encM with different combinations of homologous actinorhodin biosynthesis genes did not result in the production of oxidatively rearranged enterocin-actinorhodin hybrid compounds as anticipated, suggesting that wild-type EncM may be specific for its endogenous type II polyketide synthase or for benzoyl-primed polyketide precursors.

Plant Physiol, 2004 Nov, 136(3), 3572 - 81 Epub 2004 Oct 22.
Evidence of programmed cell death in post-phloem transport cells of the maternal pedicel tissue in developing caryopsis of maize; Kladnik A et al.; We present cellular- and ultracellular-level studies here to show developmental programmed cell death (PCD) of placento-chalazal (P-C) cell layers in maternal pedicel tissue in developing caryopses of normal seed (Mn1) and in the invertase-deficient miniature (mn1) seed mutant in maize (Zea mays) . PCD was evidenced by loss of nuclei and all subcellular membranous organizations in many P-C layers . The terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) stain that is diagnostic of apoptotic-like PCD identified spatially and temporally two distinctive subdomains, which coincided with nucellar and integumental P-C layers based on their developmental origins . The early phase of PCD in the nucellar P-C was TUNEL negative and was specific to only the fertilized caryopses, indicating that the signaling for PCD in these maternal cells originated in the zygotic tissues . In fact, the initiation of PCD coincided with endosperm cellularization and was rapidly and coordinately completed prior to the beginning of the major storage phase in endosperm . Cell shape in these cell layers was also influenced by the genotype of filial endosperm . The later phase of PCD was restricted to the integumental P-C layers underneath the nucellar cells and was TUNEL positive in both genotypes . The two subdomains of the P-C layers were also distinguishable by unique cell wall-associated phenolic compounds . Based on collective evidence, we infer that the nucellar PCD may have osmolytic etiology and may lead to activation of the post-phloem transport function of the P-C layer, whereas the integumental PCD was senescent related, in particular, protecting the maturing seed against microbes that may be transported from the maternal tissue.

Mol Biochem Parasitol, 2004 Nov, 138(1), 21 - 8
Double-stranded RNA mediates homology-dependent gene silencing of gamma-tubulin in the human parasite Entamoeba histolytica; Vayssie L et al.; Approaches that eliminate mRNA are a powerful tool for reverse genetics applications in eukaryotic microbes for which gene replacement techniques have not yet been developed . Here, for the first time, we demonstrate that RNA duplexes efficiently inhibit gene expression when introduced into the human parasite Entamoeba histolytica . Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing parasite gamma-tubulin mRNA . Use of specific antibodies revealed that microtubules and gamma-tubulin were intra-nuclear in E . histolytica . The RNAi approach to modulation of gamma-tubulin mRNA resulted in loss of the highly organized microtubule array an observation that correlates with a significant reduction of gamma-tubulin as well as of the specific mRNA . Our results suggest that gamma-tubulin is essential for microtubule nucleation in E . histolytica.

Arzneimittelforschung, 2004, 54(9), 557 - 62
A new method using flow cytometry to measure the effects of drugs on gastric emptying and gastrointestinal transit in mice; Inada T et al.; The use of radiolabelled markers is considered to be a gold standard for assessing gastric emptying and gastrointestinal transit in mice and rats, but their use has increasingly been restricted due to health concern . Therefore, a new method using fluorescent polystyrene microbeads and flow cytometry was devised . Saline containing fluorescent markers (together with non-fluorescent microbeads) was infused into the stomach of each mouse and gastric emptying and gastrointestinal transit were calculated by measuring the quantity of the fluorescent microbeads in the gastrointestinal tract using flow cytometer . The effects of saline (as a control), morphine (CAS 52-26-6) and dexmedetomidine (CAS 113775-47-6) were tested . Both gastric emptying and gastrointestinal transit measured with this method (after i.p . injection of saline) were similar to those reported previously using the conventional radiolabelled methods . Morphine significantly inhibited both gastric emptying and gastrointestinal transit in a dose-dependent manner . Dexmedetomidine did not significantly inhibit gastric emptying but inhibited gastrointestinal transit . These results were also similar to those obtained using the conventional radiolabelled method . The method using fluorescent microbeads and flow cytometry may be a reliable alternative to the methods using radioisotope for studying the effects of drugs on gastric emptying and gastrointestinal transit.

Genome, 2004 Oct, 47(5), 775 - 80
The prevalence and evolution of sex in microorganisms; Xu J; The origin of sex and how sex is maintained are among the biggest puzzles in biology . Most investigations into this problem have focused on complex eukaryotes like animals and plants . This mini-review summarizes recent progress in our understanding of the evolution of sex, highlighting results from studies of experimental and natural populations of microorganisms . Increasing evidence indicates that sexual reproduction in natural populations of viruses, bacteria, and eukaryotic microbes is much more prevalent than previously thought . In addition, investigations using experimental microbial populations are providing important parameters relevant to our understanding of the origin and maintenance of sex . It is argued that microbes are excellent model organisms to explore the mechanisms responsible for the evolution of sex.

MMWR Surveill Summ, 2004 Oct 22, 53(8), 23 - 45
Surveillance for waterborne-disease outbreaks associated with drinking water--United States, 2001-2002; Blackburn BG et al.; PROBLEM/CONDITION: Since 1971, CDC, the U.S . Environmental Protection Agency, and the Council of State and Territorial Epidemiologists have maintained a collaborative surveillance system for collecting and periodically reporting data related to occurrences and causes of waterborne-disease outbreaks (WBDOs) . This surveillance system is the primary source of data concerning the scope and effects of waterborne disease outbreaks on persons in the United States . REPORTING PERIOD COVERED: This summary includes data on WBDOs associated with drinking water that occurred during January 2001-December 2002 and on three previously unreported outbreaks that occurred during 2000 . DESCRIPTION OF SYSTEM: Public health departments in the states, territories, localities, and the Freely Associated States are primarily responsible for detecting and investigating WBDOs and voluntarily reporting them to CDC on a standard form . The surveillance system includes data for outbreaks associated with both drinking water and recreational water; only outbreaks associated with drinking water are reported in this summary . RESULTS: During 2001-2002, a total of 31 WBDOs associated with drinking water were reported by 19 states . These 31 outbreaks caused illness among an estimated 1,020 persons and were linked to seven deaths . The microbe or chemical that caused the outbreak was identified for 24 (77.4%) of the 31 outbreaks . Of the 24 identified outbreaks, 19 (79.2%) were associated with pathogens, and five (20.8%) were associated with acute chemical poisonings . Five outbreaks were caused by norovirus, five by parasites, and three by non-Legionella bacteria . All seven outbreaks involving acute gastrointestinal illness of unknown etiology were suspected of having an infectious cause . For the first time, this MMWR Surveillance Summary includes drinking water-associated outbreaks of Legionnaires disease (LD); six outbreaks of LD occurred during 2001-2002 . Of the 25 non-Legionella associated outbreaks, 23 (92.0%) were reported in systems that used groundwater sources; nine (39.1%) of these 23 groundwater outbreaks were associated with private noncommunity wells that were not regulated by EPA . INTERPRETATION: The number of drinking water-associated outbreaks decreased from 39 during 1999-2000 to 31 during 2001-2002 . Two (8.0%) outbreaks associated with surface water occurred during 2001-2002; neither was associated with consumption of untreated water . The number of outbreaks associated with groundwater sources decreased from 28 during 1999-2000 to 23 during 2001-2002; however, the proportion of such outbreaks increased from 73.7% to 92.0% . The number of outbreaks associated with untreated groundwater decreased from 17 (44.7%) during 1999-2000 to 10 (40.0%) during 2001-2002 . Outbreaks associated with private, unregulated wells remained relatively stable, although more outbreaks involving private, treated wells were reported during 2001-2002 . Because the only groundwater systems that are required to disinfect their water supplies are public systems under the influence of surface water, these findings support EPA's development of a groundwater rule that specifies when corrective action (including disinfection) is required . PUBLIC HEALTH ACTION: CDC and EPA use surveillance data 1) to identify the types of water systems, their deficiencies, and the etiologic agents associated with outbreaks and 2) to evaluate the adequacy of technologies for providing safe drinking water . Surveillance data are used also to establish research priorities, which can lead to improved water-quality regulations . CDC and EPA recently completed epidemiologic studies that assess the level of waterborne illness attributable to municipal drinking water in nonoutbreak conditions . The decrease in outbreaks in surface water systems is attributable primarily to implementation of provisions of EPA rules enacted since the late 1980s . Rules under development by EPA are expected to protect the public further from microbial contaminants while addressing risk tradeoffs of disinfection byproducts in drinking water.

Eye Contact Lens, 2004 Jul, 30(3), 163 - 5
Effectiveness of contact lens disinfectants after lens storage; Dannelly HK et al.; PURPOSE: To determine the effectiveness of multipurpose contact lens solutions after incubation with lenses for one week . METHODS: Five multipurpose solutions, three lots each, were preincubated with contact lenses for one week . The solutions were removed and one of three microbes was added to a final concentration of l x l0(6)/mL . The inoculated solutions were incubated for 4 and 6 hours; aliquots were then neutralized, serially diluted, and plated . Plate counts were used to determine the approximate number of organisms that were killed effectively by the contact lens solution . RESULTS: Of the five solutions tested, only OPTI-FREE Express maintained full effectiveness after preincubation with lenses . The other solutions were effective only with Fusarium solani after the preincubation period . CONCLUSIONS: We propose that the active component was sufficiently reduced by absorption or inactivation during storage with contact lenses such that it was rendered effective only for F . solani and was no longer concentrated enough to be effective with the bacterial species.

J Biol Chem, 2005 Jan 7, 280(1), 9 - 17 Epub 2004 Oct 21.
Surfactant Protein A Binds Mycoplasma pneumoniae with High Affinity and Attenuates Its Growth by Recognition of Disaturated Phosphatidylglycerols; Piboonpocanun S et al.; Surfactant Protein A (SP-A) is an abundant, multifunctional lectin that resides within the bronchoalveolar compartment of the lung and plays an important role in the innate immunity of the organ . Mycoplasma pneumoniae is a human pathogen that resides in the same compartment as SP-A, and we examined the interaction between the two . Preparations of human and rat SP-A recognized the mycoplasma with high affinity in the presence of Ca(2+), exhibiting apparent K(')(d) values in the nanomolar range . Membranes prepared from the microbe also bound human and rat SP-A with similar characteristics and affinity to the intact cells . The ligand for SP-A was insensitive to proteolysis . Lipid extracts prepared from the mycoplasma, bound SP-A with high affinity when examined by ligand blot analysis . These lipid extracts were also potent competitive inhibitors (IC(50) = 0.2 nm) of human SP-A binding to mycoplasma membranes . The major lipid ligands for the protein identified by mass spectrometry are a group of disaturated phosphatidylglycerols . The addition of SP-A to cultures of M . pneumoniae markedly attenuated the growth of the organism assessed by colony formation, metabolic activity, and DNA replication . The bacteriostatic effects of SP-A were reversed by dipalmitoylphosphatidylglycerol . These findings demonstrate that human SP-A can play a direct role in antibody-independent immunity to M . pneumoniae by interacting with lipid ligands expressed on the surface of the organism and implicate SP-A in the immediate host response to the bacteria.

J Pathol, 2004 Nov, 204(4), 396 - 406
Pathogen-induced actin filament rearrangement in infectious diseases; Rottner K et al.; Host defence mechanisms involve the establishment and maintenance of numerous barriers to infectious microbes, including skin and mucosal surfaces, connective tissues, and a sophisticated immune system to detect and destroy invaders . Defeating these defence mechanisms and breaching the cell membrane barrier is the ultimate challenge for most pathogens . By invading the host and, moreover, by penetrating into individual host cells, pathogens gain access to a protective niche, not only to avoid immune clearance, but also to replicate and to disseminate from cell to cell within the infected host . Many pathogens are accomplishing these challenges by exploiting the actin cytoskeleton in a highly sophisticated manner as a result of having evolved common as well as unique strategies . Copyright (c) 2004 Pathological Society of Great Britain and Ireland.

Langmuir, 2004 Oct 26, 20(22), 9769 - 74
Fabrication of microarrays on fused silica plates using the laser-induced backside wet etching method; Ding X et al.; A novel approach in the fabrication of microarrays of dye and protein on fused silica plates using the laser-induced backside wet etching (LIBWE) technique is described . The surface of fused silica plates was initially precoated using trimethoxysilane self-assembled monolayers (SAMs) and then etched using the LIBWE method to obtain the desired microstructures on the plate surface . Using this technique, the SAMs on the nonirradiated areas were able to survive the LIBWE process and were used as templates for the subsequent deposition of dye molecules or proteins via chemical bonding or physical adsorption . In the case of fused silica plates precoated with fluorinated SAMs, the LIBWE method is used to remove the SAMs to expose the etched silica surfaces, on which a thin layer of pyranine molecules can be site-selectively deposited using an aqueous solution of pyranine . In another application, an ethanol solution of rhodamine 6G was preferentially deposited onto the nonirradiated areas . In yet another application, bovine serum albumin was preferentially deposited onto the laser-irradiated areas; in this case, the fused silica plates were precoated with poly(ethylene oxide) SAMs . Interestingly, when an aqueous suspension of polystyrene (PS) microbeads was cast onto the fused silica precoated with the fluorinated SAMs, hexagonally close-packed PS microbeads were deposited into the etched cavities . Depositions of the dye, protein, and microbeads were confirmed by visualization using a fluorescence microscope and scanning electron microscope.

Springer Semin Immunopathol . 2004 Oct 14; {Epub ahead of print}
Natural endogenous adjuvants; Rock KL et al.; It 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.

Med Hypotheses, 2004, 63(5), 773 - 7
Recycling of pathogenic microbes through survival in ice; Rogers SO et al.; Viable microorganisms (e.g . fungi, bacteria, Archaea and viruses) are distributed by wind over great distances, including globally . Microbes may settle out of the atmosphere or may be incorporated into fog, rain, sleet, hail, or snow . These organisms fall into lakes, streams, oceans, or onto the land or glaciers . When they become incorporated into environmental ice (e.g . glaciers, ice sheets, and snow), those that survive freezing and thawing may persist for years, centuries, millennia, or longer . Once they melt from the ice, they may enter contemporary populations . This mixing of ancient and modern genotypes (i.e . temporal gene flow, or what we term "genome recycling") may lead to a change of allele proportions in the population, which may have effects on mutation rates, fitness, survival, pathogenicity and other characteristics of the organisms . Pathogenic microbes that survive freezing and thawing (e.g . influenza viruses, polioviruses, caliciviruses and tobamoviruses) can remain in these icy reservoirs long enough to avoid resistance mechanisms of the hosts, thereby conveying a selective advantage to these pathogens over those that cannot survive in ice . Ice is an abiotic reservoir of microbes that has been ignored in surveillance activities for human diseases.

Annu Rev Microbiol, 2004, 58, 207 - 31
The ecology and genetics of microbial diversity; Kassen R et al.; Natural communities of microbes are often diverse, a fact that is difficult to reconcile with the action of natural selection in simple, uniform environments . We suggest that this apparent paradox may be resolved by considering the origin and fate of diversity in an explicitly ecological context . Here, we review insights into the ecological and genetic causes of diversity that stem from experiments with microbial populations evolving in the defined conditions of the laboratory environment . These studies highlight the importance of environmental structure in governing the fate of diversity and shed light on the genetic mechanisms generating diversity . We conclude by emphasizing the importance of placing detailed molecular-level studies within the context of a sound ecological and evolutionary framework.

Expert Rev Anti Infect Ther, 2003 Dec, 1(4), 597 - 608
Pathogenesis of Guillain-Barre syndrome; Tsang RS et al.; Guillain-Barre syndrome is a postinfectious disorder caused by an aberrant immune response to an infectious pathogen, resulting in an autoimmune disease . As with other autoimmune diseases of infectious nature, the intricate balance of the numerous factors involved in the immune response may determine the outcome of the interaction between the microbe and host . Recent studies focusing on the role of cytokines and its network of related mediators and receptors suggest that any imbalance may make a significant contribution to the outcome of the infectious disease process . Better understanding of the pathogenesis of Guillain-Barre syndrome may lead to the discovery of newer therapeutics and may also serve as a model for studying other autoimmune diseases.

Appl Spectrosc, 2004 Sep, 58(9), 1051 - 6
Assessing soil respiration by means of near-infrared diode laser spectroscopy; Gianfrani L et al.; High-resolution diode laser spectroscopy in the near-infrared region is applied to the accurate measurement of soil respiration . In particular, the use of a diode-laser-based spectrometer has allowed the implementation, for the first time, of a static accumulation method capable of measuring soil respiration from continuous measurements of CO(2) concentrations, with minor perturbation on soil respiration as well as on CO(2) transport and emission . The system has been tested in a laboratory experiment by detection of CO(2) production from sandy matrices, inoculated with active soil microbes and supplied with different amounts of decomposable plant material . Respiration rates of all samples were then retrieved using a diffusion model . The results of the laboratory tests are in agreement with those expected on the basis of sample composition . Examples of operation with real soil samples are also reported . We discuss the possible field application of the system, in conjunction with closed static soil chambers.

Environ Microbiol, 2004 Nov, 6(11), 1137 - 48
Widespread association of a Rickettsiales-like bacterium with reef-building corals; Casas V et al.; White band disease type I (WBD I) has been a major cause of the dramatic decline of Acroporid coral populations throughout the Caribbean during the last two decades, yet the aetiological agent of this disease is unknown . In this study, the bacterial communities associated with both healthy and diseased Acropora species were compared by 16S rDNA analyses . The bacterial communities of both healthy and diseased Acropora spp . were dominated by a single ribotype with 90% identity to a bacterium in the order Rickettsiales . Screening by nested PCR specific to the coral-associated Rickettsiales 1 (CAR1) bacterium showed that this microbe was widespread in both healthy and diseased A . cervicornis and A . palmata corals from 'healthy' (i.e . low WBD I incidence) and 'stressed' reefs (i.e . high WBD I incidence) . These results indicate that there were no dramatic changes in the composition of the microbial community associated with WBD I . CAR1 was also associated with non-Acroporid corals of the Caribbean, as well as with two Acroporid corals native to the Pacific . CAR1 was not present in the water column . This bacterium was also absent from preserved Caribbean Acroporid samples collected between 1937 and 1980 before the outbreak of WBD I . These results suggest CAR1 is a relatively new bacterial associate of Acroporids and that a non-bacterial pathogen might be the cause of WBD I.

Mol Immunol, 2004 Nov, 41(11), 1099 - 108
The role of Toll-like receptors and Nod proteins in bacterial infection; Philpott DJ et al.; Our understanding of innate immunity in mammals has greatly expanded following the discovery of the family of membrane-bound receptors, called the Toll-like receptors (TLRs) . More recently, the nucleotide-binding oligomerisation domain (Nod) molecules, Nod1 and Nod2, which are cytoplasmic surveillance proteins, have also been shown to be involved in the innate immune response . These two classes of detection molecules, classified as "pattern recognition receptors" (PRRs), detect microbial ligands in order to initiate a defense response to fight infectious disease . These microbial ligands or "pathogen-associated molecular patterns" (PAMPs), detected by TLRs and Nods are often structural components of the microorganism that are not subject to much variation . These include such factors as lipopolysaccharide (LPS) and peptidoglycan from the cell walls of bacteria . In order to understand the role of TLRs and Nod proteins in infectious disease in vivo it is important to define the site of interaction between PRRs and PAMPS . Additionally, the challenge of mice deficient in the various PRRs in natural infection models will help to decipher the contribution of these molecules not only in the innate immune response against pathogen infection but also how these proteins may instruct the adaptive immune response in order to have a tailored immune response against a particular microbe.

Gene, 2004 Oct 13, 340(2), 267 - 74
Structure and function of a cellulase gene in redclaw crayfish, Cherax quadricarinatus; Crawford AC et al.; The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes . Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al . {Gene 239 (1999) 317-324.} . Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene . Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase . Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes . The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2) . Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region . Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene . These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes.

Scanning, 2004 Jul-Aug, 26(4), 175 - 80
A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy; Stadtlander CT et al.; This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy . Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe . These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures . We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide . Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas . Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.

Clin Infect Dis, 2004 Sep 15, 39(6), 827 - 30 Epub 2004 Aug 27.
When microbe meets human; Reid G; Microbes make up a significant component of the human body, yet relatively little is known about how they influence health and disease . They colonize after birth by chance and circumstance, yet play a major role in immunity, digestion, and protection against disease . In relatively recent times, basic science and clinical studies have clearly shown the potential impact of indigenous and exogenous microbes on human health and well-being . Yet regulatory bodies, research funding agencies, and health care practitioners, perhaps disillusioned by too many unreliable, overhyped products that are marketed under the guise of probiotics or natural therapeutics, have lagged far behind in embracing this avenue of enquiry . As more scientifically proven probiotic products differentiate themselves from untested and unproven cure-alls, and as multidisciplinary research groups piece together the diverse components of the puzzle, humans will slowly begin to understand how best to optimize their coexistence with microbial organisms, thus perhaps prolonging and enhancing life.

Nature, 2004 Oct 28, 431(7012), 1092 - 5 Epub 2004 Oct 06.
Bioturbators enhance ecosystem function through complex biogeochemical interactions; Lohrer AM et al.; Predicting the consequences of species loss is critically important, given present threats to biological diversity such as habitat destruction, overharvesting and climate change . Several empirical studies have reported decreased ecosystem performance (for example, primary productivity) coincident with decreased biodiversity, although the relative influence of biotic effects and confounding abiotic factors has been vigorously debated . Whereas several investigations focused on single trophic levels (for example, grassland plants), studies of whole systems have revealed multiple layers of feedbacks, hidden drivers and emergent properties, making the consequences of species loss more difficult to predict . Here we report functionally important organisms and considerable biocomplexity in a sedimentary seafloor habitat, one of Earth's most widespread ecosystems . Experimental field measurements demonstrate how the abundance of spatangoid urchins--infaunal (in seafloor sediment) grazers/deposit feeders--is positively related to primary production, as their activities change nutrient fluxes and improve conditions for production by microphytobenthos (sedimentatry microbes and unicellular algae) . Declines of spatangoid urchins after trawling are well documented, and our research linking these bioturbators to important benthic-pelagic fluxes highlights potential ramifications for productivity in coastal oceans.

Mol Microbiol, 2004 Oct, 54(2), 464 - 77
Characterization of a lipoprotein, NilC, required by Xenorhabdus nematophila for mutualism with its nematode host; Cowles CE et al.; Xenorhabdus nematophila is a gamma-proteobacterial mutualist of an insect-pathogenic nematode, Steinernema carpocapsae . X . nematophila requires nilC, a gene predicted to encode an outer membrane lipoprotein of unknown function, for colonization of its nematode host . Characterization of NilC, described here, demonstrated it is a 28 kDa lipoprotein directed to the periplasm by an N-terminal signal sequence . Lipidation and processing of NilC occurs by a mechanism that is conserved in proteobacteria . This work also showed NilC is membrane associated and oriented towards the periplasm of X . nematophila and is produced as an outer membrane-associated protein when expressed in Escherichia coli . Expression analyses revealed that nilC transcription is directly or indirectly repressed by Lrp, and this regulatory link may explain the nematode mutualism defect of a previously identified lrp::Tn5 mutant . An lrp::Tn5 mutant produces an additional nilC transcript, not observed in wild-type cells growing in vitro, and produces approximately 75-fold more nilC than wild-type cells in late stationary phase . These fundamental characterizations of nilC expression and nilC localization and processing events have provided firm bases for understanding the role of this colonization factor in the X . nematophila/S . carpocapsae microbe-host interaction.

Nat Genet, 2004 Nov, 36(11), 1165 - 73 Epub 2004 Oct 03.
Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity; Cazalet C et al.; Legionella pneumophila, the causative agent of Legionnaires' disease, replicates as an intracellular parasite of amoebae and persists in the environment as a free-living microbe . Here we have analyzed the complete genome sequences of L . pneumophila Paris (3,503,610 bp, 3,077 genes), an endemic strain that is predominant in France, and Lens (3,345,687 bp, 2,932 genes), an epidemic strain responsible for a major outbreak of disease in France . The L . pneumophila genomes show marked plasticity, with three different plasmids and with about 13% of the sequence differing between the two strains . Only strain Paris contains a type V secretion system, and its Lvh type IV secretion system is encoded by a 36-kb region that is either carried on a multicopy plasmid or integrated into the chromosome . Genetic mobility may enhance the versatility of L . pneumophila . Numerous genes encode eukaryotic-like proteins or motifs that are predicted to modulate host cell functions to the pathogen's advantage . The genome thus reflects the history and lifestyle of L . pneumophila, a human pathogen of macrophages that coevolved with fresh-water amoebae.

Appl Environ Microbiol, 2004 Oct, 70(10), 6282 - 9
Abundance of reverse tricarboxylic acid cycle genes in free-living microorganisms at deep-sea hydrothermal vents; Campbell BJ et al.; Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem . However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of Proteobacteria and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate . Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents . PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples . These genes were also expressed in at least one chimney sample . Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of Proteobacteria . These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of Proteobacteria in free-living hydrothermal vent communities . Members of the epsilon subdivision of Proteobacteria are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface . Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.

Appl Environ Microbiol, 2004 Oct, 70(10), 5842 - 6
Movement of viruses between biomes; Sano E et al.; Viruses are abundant in all known ecosystems . In the present study, we tested the possibility that viruses from one biome can successfully propagate in another . Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil . The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-microm-pore-size filter) . Virus-like particle abundances were then monitored using direct counting . Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate . These results imply that viruses can laterally transfer DNA between microbes in different biomes.

Curr Issues Intest Microbiol, 2004 Sep, 5(2), 31 - 47
Molecular microbial ecology of the gastrointestinal tract: from phylogeny to function; Zoetendal EG et al.; During the past decade it became evident that anaerobic cultivation-based approaches provides an incomplete picture of the microbial diversity in the GI tract, since at present only a minority of microbes can be obtained in culture . The application of molecular, mainly 16S ribosomal RNA (rRNA)-based approaches enables researchers to bypass the cultivation step and has proven its usefulness in studying the microbial composition in a variety of ecosystems, including the gastrointestinal (GI) tract . This critical review summarizes the impact of these culture-independent approaches on our knowledge of the ecology of the GI tract and provides directions for future studies which should emphasize function of specific strains, species and groups of microbes.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1490 - 5
Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase; Roy I et al.; Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution . The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively . Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format . The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation . The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

Biophys J, 2004 Dec, 87(6), 4203 - 12 Epub 2004 Dec.
Controlled ablation of microtubules using a picosecond laser; Botvinick EL et al.; The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies . In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex . Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule . Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the alpha-subunit of tubulin . Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle . Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms . We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs . The possible photon interaction mechanisms are discussed in detail.

Best Pract Res Clin Rheumatol, 2004 Oct, 18(5), 689 - 703
Reactive arthritis; Toivanen A et al.; Reactive arthritis is an infectious disease which may be initiated by several microbes in genetically susceptible hosts . The best known predisposing genetic factor is HLA-B27, but the mechanisms behind its action are still elusive . Worldwide agreement exists regarding the general guidelines in the diagnosis, differential diagnosis and management, even though official diagnostic criteria are not yet available . Several studies indicate that antibiotics are effective only if started before the immunological mechanisms of pathogenesis have been turned on . However, recent observations suggest that a 3-month course of antibiotics may diminish the late risk of chronic sequelae, especially in HLA-B27-positive patients with reactive arthritis.

J Exp Med, 2004 Oct 4, 200(7), 905 - 16 Epub 2004 Sep 27.
Chlamydia inhibit host cell apoptosis by degradation of proapoptotic BH3-only proteins; Fischer SF et al.; Chlamydia are obligate intracellular bacteria that replicate in a vacuole inside a host cell . Chlamydial infection has been shown to protect the host cell against apoptotic stimuli . This is likely important for the ability of Chlamydia to reproduce in human cells . Here we show that resistance to apoptosis is conveyed by the destruction of the proapoptotic BH3-only proteins Bim/Bod, Puma, and Bad during infection . Apoptotic stimuli were blocked upstream of the mitochondrial activation of Bax/Bak . During infection with both species, Chlamydia trachomatis and Chlamydia pneumoniae, Bim protein gradually disappeared without noticeable changes in Bim mRNA . The disappearance was blocked by inhibitors of the proteasome . Infected cells retained sensitivity to Bim expressed by transfection, indicating functional relevance of the Bim disappearance . Fusion to Bim targeted the green fluorescent protein for destruction during infection . Analysis of truncation mutants showed that a short region of Bim containing the BH3 domain was sufficient for destruction during chlamydial infection . Like Bim, Puma and Bad proteins disappeared during infection . These results reveal a novel way by which microbes can interfere with the host cell's apoptotic machinery, and provide a molecular explanation of the cellular resistance to apoptosis during infection with Chlamydia.

J Cell Biol, 2004 Sep 27, 166(7), 1041 - 54
Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway; Esselens C et al.; Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C . Esselens, V . Baert, C . Boeve, G . Snellings, P . Cupers, K . Craessaerts, and B . De Strooper . 2001 . Neuron . 32:579-589) . Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons . TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3 . Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity . Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles . TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations . Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation . Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.

Curr Opin Microbiol, 2004 Oct, 7(5), 492 - 8
Metagenomics--the key to the uncultured microbes; Streit WR et al.; It is widely accepted that up to 99.8% of the microbes present in many environments are not readily culturable . 'Metagenome technology' tries to overcome this bottleneck by developing and using culture-independent approaches . From the outset, metagenome-based approaches have led to the accumulation of an increasing number of DNA sequences, but until this time the sequences retrieved have been those of uncultured microbes . These genomic sequences are currently exploited for novel biotechnological and pharmaceutical applications and to increase our knowledge on microbial ecology and physiology of these microbes . Using the metagenome sequences to fully understand how complex microbial communities function and how microbes interact within these niches represents a major challenge for microbiologists today.

Infect Genet Evol, 2004 Sep, 4(3), 221 - 42
Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases; Keys CJ et al.; A database of MALDI-TOF mass spectrometry (MS) profiles has been developed with the aim of establishing a high throughput system for the characterisation of microbes . Several parameters likely to affect the reproducibility of the mass spectrum of a taxon were exhaustively studied . These included such criteria as sample preparation, growth phase, culture conditions, sample storage, mass range of ions, reproducibility between instruments and the methodology prior to database entry . Replicates of 12 spectra per sample were analysed using a 96-well target plate containing central wells for peptide standards to correct against mass drift during analysis . The quality of the data was assessed statistically prior to database addition using root mean squared values of <3.0 as the criterion for rejection . Cluster analysis using a nearest neighbour algorithm also enabled subsets of data to be compared . This was achieved using the bespoke MicrobeLynx trade mark software . Columbia blood agar was used to standardise all procedures for the database, since it permitted the culture of most human pathogens and also produced spectra with a broad range of mass ions . In some instances, alternative media such as CLED were used in specific studies with greater success . Following standardisation of the procedure, a database was developed comprising ca . 3500 spectra with multiple strain entries for most species . The results to date show unequivocally that as the number of strains per species increased, so too did the success of species matching . The technique demonstrated unique mass spectral profiles for each genus/species, with the variation in mass ions among strains/species being dependent on the intra-specific diversity . The success of identification against the database for wild-type strains ranged between 33 and 100%; the lower percentage results being generally associated with poor representation of some species within the database . These findings provide a new dimension for the rapid and high throughput characterisation of human pathogens with potentially broad applications across the field of microbiology.

Osiris, 2004, 19, 39 - 61
Natural histories of infectious disease: ecological vision in twentieth-century biomedical science; Anderson W; During the twentieth century, disease ecology emerged as a distinct disciplinary network within infectious diseases research . The key figures were Theobald Smith, F . Macfarlane Burnet, Rene Dubos, and Frank Fenner . They all drew on Darwinian evolutionism to fashion an integrative (but rarely holistic) understanding of disease processes, distinguishing themselves from reductionist "chemists" and mere "microbe hunters." They sought a more complex, biologically informed epidemiology . Their emphasis on competition and mutualism in the animated environment differed from the physical determinism that prevailed in much medical geography and environmental health research . Disease ecology derived in part from studies of the interaction of organisms - micro and macro - in tropical medicine, veterinary pathology, and immunology . It developed in postcolonial settler societies . Once a minority interest, disease ecology has attracted more attention since the 1980s for its explanations of disease emergence, antibiotic resistance, bioterrorism, and the health impacts of climate change.

Radiat Res, 2004 Oct, 162(4), 426 - 32
Biological responses in known bystander cells relative to known microbeam-irradiated cells; Ponnaiya B et al.; Normal human fibroblasts in plateau phase ( congruent with 95% G(1) phase) were stained with the vital nuclear dye Hoechst 33342 (blue fluorescence) or the vital cytoplasmic dye Cell Tracker Orange (orange fluorescence) and plated at a ratio of 1:1 . Only the blue-fluorescing nuclei were microbeam-irradiated with a defined number of 90 keV/microm alpha particles . The orange-fluorescing cells were then "bystanders", i.e . not themselves hit but adjacent to cells that were . Hit cells showed a fluence-dependent induction of micronuclei as well as delays in progression from G(1) to S phase . Known bystander cells also showed enhanced frequencies of micronuclei (intermediate between those seen in irradiated and control cells) and transient cell cycle delays . However, the induction of micronuclei in bystander cells did not appear to be dependent on the fluence of the particles delivered to the neighboring hit cells . These are the first studies in which the bystander effect has been visualized directly rather than inferred . They indicate that the phenomenon has a quantitative basis and imply that the target for radiation effects cannot be considered to be the individual cell.

Radiat Res, 2004 Oct, 162(4), 474 - 9
Microdosimetry of electron microbeams; Miller JH et al.; Track structures of 25, 50 and 80 keV primary electrons, simulated by the detailed-history Monte Carlo method, were analyzed for the frequency distributions of energy deposited in spheres with a diameter of 1 microm, placed in a cylindrically symmetrical array around the projected initial direction of the primary electron . The frequency mean of specific energy, the dose mean of lineal energy, and the parameters of lognormal functions fit to the dose distributions were calculated as a function of beam penetration and radial distance from the projected beam axis . Given these data, the stochastics of dose and radiation quality for micrometer-scale sites targeted by a medium-energy electron microbeam can be predicted as a function of the site's location relative to the beam entry point.

Anesth Analg, 2004 Oct, 99(4), 1102 - 6, table of contents
Propofol and midazolam inhibit gastric emptying and gastrointestinal transit in mice; Inada T et al.; We studied the effect of propofol and midazolam on gastric emptying and gastrointestinal transit in mice . Ten minutes after intraperitoneal injection of propofol or midazolam, 0.2 mL of saline containing fluorescent microbeads was infused into the stomach . Thirty minutes later, the gastrointestinal tract was excised, and gastric emptying and gastrointestinal transit were calculated by measuring the quantity of fluorescent microbeads in the gastrointestinal tract by using a flow cytometer . At a dose that produced a light level of sedation (mice righted themselves within 2 s), both drugs significantly, but weakly, inhibited gastric emptying to a similar degree (propofol: P < 0.001 versus control value; 95% confidence interval {CI} for difference, 4.9%-20.2%; midazolam: P < 0.001 versus control value; 95% CI for difference, 7.8%-14.7%) . Midazolam, but not propofol, delayed gastrointestinal transit (P < 0.001) . At a larger dose that produced a deeper level of sedation (absence of righting reflex >10 s), both drugs significantly inhibited gastric emptying (propofol: P < 0.001; 95% CI for difference, 31.4%-61.2%; midazolam: P < 0.001; 95% CI for difference, 30.8%-61.1%) and gastrointestinal transit (P < 0.001 for both drugs).

J Biol Chem, 2004 Nov 26, 279(48), 49910 - 8 Epub 2004 Nov 26.
Regulation of anaerobic dehalorespiration by the transcriptional activator CprK; Pop SM et al.; Desulfomonile, Desulfitobacterium, and Dehalobacter are anaerobic microbes that can derive energy from the reductive dehalogenation of chlorinated organic compounds, many of which are environmental pollutants . There is very little information about how anaerobic dehalorespiration is regulated . An open reading frame within the Desulfitobacterium dehalogenans chlorophenol reductase (cpr) gene cluster (cprK) was proposed to be a transcriptional regulatory protein (Smidt, H., van Leest, M., van der Oost, J., and deVos, W . M . (2000) J . Bacteriol . 182, 5683-5691) . We have cloned, actively overexpressed in Escherichia coli, and purified to homogeneity the D . dehalogenans CprK . The results of electrophoretic mobility shift assays, DNA footprinting studies, and promoter-lac fusion experiments indicate that CprK is a transcriptional activator of the cpr gene cluster . CprK binds 3-chloro-4-hydroxyphenylacetate (CHPA) with high affinity (K(d) = 3.5 mum, determined by isothermal titration calorimetry), which promotes its specific interaction with a DNA sequence (TTAAT-N4-ACTAA) located upstream of the -35 and -10 promoter regions of several cpr genes and activates transcription of these genes . Binding to the upstream "box" sequence increases the affinity of CprK for CHPA by approximately 10-fold (K(d) = 0.4 mum, determined by electrophoretic mobility shift assays) . Chlorophenylacetate, which lacks the ortho-hydroxy group, and hydroxyphenylacetate, lacking the chlorine group, do not activate transcription or promote DNA binding, even at millimolar concentrations, at least 1000-fold higher than the K(d) value for CHPA . Lacking metals, CprK is oxygen-sensitive . Oxidation by diamide, which converts thiols to the disulfide, inactivates CprK, and reduction of the oxidized protein by dithiothreitol fully restores DNA binding, indicating that CprK is redox-regulated and is active only when reduced . This is the first reported characterization of a transcriptional regulator of anaerobic dehalorespiration.

J Anim Physiol Anim Nutr (Berl), 2002 Aug, 86(7-8), 239 - 45
Absorption and metabolism of alpha-ketoglutarate in growing pigs; Kristensen NB et al.; The portal appearance of enteral alpha-ketoglutarate (AKG) and the effect of enteral or parenteral AKG on portal net appearance of glucose, short-chain fatty acids, alanine, aspartate, glutamate, glutamine, proline and insulin were investigated in three growing pigs . During the experimental samplings the pigs were fed hourly with a standard feed mix with 5% glucose (control), 5% AKG (enteral) or no feed additive but continuously infused with AKG into the mesenteric vein in an amount equivalent to 5% of feed intake (parenteral) . The arterial plasma concentration of AKG increased (p < 0.05) following both enteral (from 16+/-2 to 22+/-3 micromol/l) and parenteral (from 16+/-2 to 425+/-27 micromol/l) administration of AKG . With the enteral treatment 4+/-1% of the AKG could be accounted for in the portal vein, however, with the parenteral treatment 86+/-5% could be accounted for in the portal vein . The arterial plasma concentration of proline increased (p < 0.05) with the enteral treatment (365 +/- 3 to 443 +/- 39 micromol/l), but was not affected by the parenteral treatment (p > 0.10) . The plasma concentration glutamine decreased (p < 0.05) with the parenteral treatment only . The portal net appearance of proline showed a numerical increase with the enteral treatment but no other affects on arterial concentrations or portal net appearance were found . A small accompanying study showed that only small amounts of enteral AKG was present in the small intestine . It was therefore concluded that enteral AKG has a low availability to peripheral tissues either because it is absorbed and metabolized in the stomach and duodenum or because it is metabolized by microbes in the stomach . The study showed that AKG is metabolized differently following enteral and parenteral application in growing pigs.

Biomed Microdevices, 2004 Sep, 6(3), 213 - 8
A microfluidic device for electrofusion of biological vesicles; Tresset G et al.; This paper reports a microfabricated device with high aspect-ratio electrodes and low power consumption for the electrofusion of liposomes and cells . The applications may range from gene transfection or cell tracking to biophysical studies of membrane proteins . The device consists of 250 microm thick silicon electrodes bonded to a glass substrate and covered by a PDMS-coated glass slide . Liposomes were first aligned by AC voltage at 300 kHz and then fused with short DC pulses . The fusion yield can reach 75% and is globally better for liposome diameters larger than 10 microm . The encapsulation of microbeads inside liposomes has also been demonstrated and opens up the route towards fusion-based delivery of artificial microstructures into cells.

IEEE Trans Nanobioscience, 2003 Dec, 2(4), 247 - 54
Effect of magnetic bead agglomeration on Cytomagnetometric measurements; Moller W et al.; Magnetic twisting cytometry (MTC) is a novel tool to measure cytoskeleton-associated cell functions by the use of ferromagnetic microbeads . Magnetic beads are either incorporated by living cells by phagocytic processes or attached to integrin receptors to the cell membrane . The magnetic beads are magnetized and aligned in a strong magnetic field pulse . The application of twisting forces allows to investigate mechanical properties (stiffness, viscoelasticity) of the cytoskeleton of living cells by analyzing the magnetic cell field . Incorporated magnetic beads undergo intracellular transport processes, which result in a loss of particle alignment and in a decay of the remanent magnetic cell field . This process, called relaxation, depends on the mechanical cytoskeletal properties and can directly visualize the intracellular energy of cellular transport processes . The preparation of spherical monodisperse ferromagnetic beads made it possible to understand the above-described processes using mathematical models . Experimental conditions with many magnetic particles per cell enhances the formation of aggregates because of the attractive forces between magnetic spheres, resulting in a change of magnetic properties and of hydrodynamic behavior . Due to mutual magnetization, the remanent magnetic moment of an aggregate is stronger compared to the same number of single particles . This implies a higher cell field . Additionally the relaxation is retarded because of the change in shape factor and in volume, which also implies a faulty estimation of intracellular transport energy . Magnetic particle twisting is less influenced . In summary, valuable cytomagnetometric measurements have to be done with less than one particle per macrophage to ensure low probability of multiple particles per cell.

Int J Oncol, 2004 Oct, 25(4), 797 - 819
Genome-wide gene-expression profiles of breast-cancer cells purified with laser microbeam microdissection: identification of genes associated with progression and metastasis; Nishidate T et al.; Breast carcinoma is a complex disease characterized by accumulation of multiple genetic alterations, and the understanding of the molecular basis of mammary tumorigenesis is still incomplete . In this study we analyzed gene-expression profiles of 81 surgical specimens of 12 ductal carcinoma in situ (DCIS) and 69 invasive ductal carcinoma (IDC) . After applying laser-microbeam micro-dissection to all samples we achieved 98-99% pure populations of breast cancer cells, and of normal breast epithelial cells used as controls . A cDNA-microarray analysis of 23,040 genes in these samples and a subsequent unsupervised hierarchical clustering distinguished two tumor groups, mainly in terms of estrogen-receptor (ER) status . We then undertook a supervised analysis and identified 325 genes that were commonly either up- or down-regulated in both pathologically discrete stages (DCIS and IDC), indicating that these genes might play important roles in malignant transformation of breast ductal cells . In addition, we searched invasion-associated gene candidates whose expression was altered in IDC, but not in DCIS, and identified 24 up-regulated genes and 41 down-regulated genes . Furthermore, we identified 34 genes that were expressed differently in tumors from patients with lymph node metastasis as opposed to no metastasis . On that basis we developed a scoring system that correlated well with the metastatic status . Tumors from all of the 37 test patients with lymph-node metastasis yielded positive scores by our definition, whereas 38 of the 40 tumors (95%) without lymph node metastasis had negative scores . Our data should provide useful information for identifying predictive markers for invasion or metastasis, and suggest potential target molecules for treatment of breast cancers.

Clin Microbiol Infect, 2004 Oct, 10(10), 948 - 50
MOP-UP: an online tool for finding strain-specific primers or motifs in DNA or protein alignments; Underwood AP et al.; MOP-UP is a web-based application that enables efficient searching of nucleic acid or amino-acid alignments for sequences or motifs that are unique to a subset of the members represented in the alignment . This has applications in the design of assays that aim to detect particular strains or species . Since molecular-based characterisation of microbes is becoming increasingly important, MOP-UP can aid microbiologists in finding the best loci on which to base such assays . The program is accessible at: http://www.hpa.org.uk/srmd/bioinformatics/tools/mop-ups.htm.

Anal Chem, 2004 Sep 1, 76(17), 4960 - 7
Electrochemical and bioelectrochemistry properties of room-temperature ionic liquids and carbon composite materials; Zhao F et al.; Room-temperature ionic liquids (RTILs) are liquids at room temperature and represent a new class of nonaqueous but polar solvents with high ionic conductivity . The conductivity property of carbon nanotubes/RTILs and carbon microbeads/RTILs composite materials has been studied using ac impedance technology . Enzyme coated by RTILs-modified gold and glassy carbon electrodes allow efficient electron transfer between the electrode and the protein and also catalyze the reduction of O2 and H2O2 .

Can J Gastroenterol, 2004 Aug, 18(8), 493 - 500
Bacterial colonization and the development of intestinal defences; Shi HN et al.; In humans, intestinal defences develop during gestation and, at full term, have the capacity to respond in an appropriate manner to infectious agents and foreign antigens . Before an active protective response can occur, however, the gut must first be exposed to colonizing bacteria . Colonization with diverse intestinal microbes is necessary for the development of important gut defenses such as the synthesis and secretion of polymeric immunoglobulin A and the generation of a balanced T helper (Th) cell response . Insights into normal immune physiological development of the gut have been made by studying the germ-free animal and intestinal defenses . These studies have provided insights into the physiology of immune responses . Two important immunological functions are the secretion of polymeric immunoglobulin A to protect the intestinal surface against harmful stimuli and inhibition of the systemic response to commensal bacteria and food proteins (eg, oral tolerance) to prevent chronic inflammation . Neither function exists in the germ-free state, but rapidly develops after conventionalization (colonization) of the germ-free animal . In the present review, the importance of bacterial colonization on the appearance of normal mucosal immune function and to the clinical consequences of inadequate colonization to the development of disease will be discussed . For example, excessive Th2 activity can lead to atopy, whereas Th1 predominance is found in conditions such as Helicobacter pylori gastritis and Crohn's disease . With the eradication of infectious diseases in developed countries in the past three decades, the incidence of atopic and autoimmune diseases has increased . This epidemiological observation has been explained by the 'hygiene hypothesis', which suggests that a reduction in microbial burden by public health measures has contributed to an immunological imbalance in the intestine . A family of pattern recognition receptors (Toll-like receptors) on gut lymphoid and epithelial cells mediates innate immune responses to bacterial molecular patterns and, thereby, orchestrates acquired immunity . As the role of bacterial communication within the gut (bacterial-epithelial cross-talk) is clarified, physicians should be able to modulate gut immune responses, for example, by the use of probiotics.

J Microbiol Methods, 2004 Nov, 59(2), 223 - 31
RNA fingerprinting--a new method to screen for differences in plant litter degrading microbial communities; Aneja MK et al.; Microbial activities are essential for the nutrient turnover processes in soil and play an important role in the degradation of complex organic material, for example, plant leaf litter . However, very little is known about the microorganisms and their genes involved during the course of leaf litter decomposition . In the present study, we describe the non-radioactive application of RNA arbitrarily primed-PCR (RAP-PCR) protocol in combination with the classic litter bag technique to investigate the metabolic profiles of microbial community involved in leaf litter degradation after 2 and 8 weeks of degradation in four different soil sites, without using selective primer systems for PCR . Due to the significantly reduced target sites for PCR primers, compared to the published papers about RAP fingerprinting of more complex microbial communities based on DNA analysis (only transcripts from microbes on the litter material were analysed), the patterns of parallel samples were highly reproducible (>95%) . Shifts in microbial community structure and function were observed during the course of degradation . Each litter sample had its unique metabolic profile and both soil effects and litter quality effects were evident . RAP-PCR products were also cloned to generate libraries . Clone libraries were screened by restriction fragment length polymorphism (RFLP) and representative samples sequenced to identify the inserts . Both mRNA and rRNA transcripts were obtained confirming the presence of mRNA in total RNA preparations . Hence, the described protocol is a good screening method to find similarities or differences in the structure and function of microbial communities involved in litter degradation, which may be the basis for more detailed studies by cloning and sequencing approaches.

Rev Environ Contam Toxicol, 2004, 183, 99 - 113
Toxicity and residues of endosulfan isomers; Sutherland TD et al.; The continued availability of endosulfan is desirable for the production of cotton, and various other crops, because of its particular suitability for use in IPM and resistance management strategies . However, ongoing residue problems threaten the availability of the insecticide . Data described here suggest a beta-enriched insecticide is worthy of investigation as a useful alternative organochloride insecticide, having the advantages of reduced environmental and health concerns . The alpha-isomer of endosulfan is an order of magnitude more volatile than the beta-isomer, which is reflected in its initial rapid disappearance in the field . Approximately 70% of endosulfan is lost within 2 d of application due to volatilization . Based on volatilization kinetics, the volatilization of a beta-endosulfan formulation would be less than 5% that of commercial endosulfan . However, while it has been established that endosulfan vapor does lead to contamination of the riverine environment, this contamination is below residue levels recorded in rivers during the cotton growing season and, as such, reducing the volatility of the insecticide will only partially alleviate residue problems . Initial field trial results suggest that beta-endosulfan insecticide can achieve equivalent efficacy to commercial endosulfan at half the recommended label application rate; presumably this is a reflection of its containment on site in comparison to the more volatile commercial mix of isomers . An insecticide composed primarily of the beta-isomer would have reduced volatility and equivalent efficacy at lower application rates compared to the commercial mix of isomers, reducing offsite endosulfan residues . An important advantage of a beta-enriched insecticide would be its potential to minimize endosulfan residues in locally grown production animals . The predominant endosulfan residue in animal fat is endosulfate, accumulated after the animal ingests the metabolite while grazing on pastures contaminated by endosulfan spray drift . As the beta-isomer is oxidized on the surface of plants and by microbes at much lower rates than the alpha-isomer, endosulfate levels would be lower as a result of a contamination event with a beta-endosulfan-based insecticide compared to the commercial mix . Finally, acute toxicity against mammals of the alpha-isomer is more than three times that of the beta-isomer, and the neurotoxicity of the insecticide has been attributed to the alpha-isomer . Therefore, a beta-enriched insecticide will be less acutely and chronically toxic to agricultural workers than the commercially available insecticide . In conclusion, these properties suggest that the alpha-isomer contributes more significantly to the residue problems associated with the insecticide than the beta-isomer and that the use of a beta-isomer-based insecticide would reduce residue problems yet retain the advantages to IPM and resistant management strategies unique to the current endosulfan formulation.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Sep, 20(5), 629 - 31
{A novel microbeads-based chemiluminescence immunoassay for detecting human thyrotropin}; Wang YN et al.; AIM:To establish a low cost and sensitive microbeads-based chemiluminescence immunoassay (CLEIA) for detecting human thyrotropin . METHODS: Thyrotropin in sera was captured by an alkaline phosphase-labeled mAb against TSH beta subunit and a FITC-labeled mAb against TSH alpha subunit . Captured thyrotropin was then isolated with immuno-magnetic beads conjugated with anti-FITC antibody and then quantified by chemiluminescence using adamanatane amine as luminescent substrate . RESULTS: The sensitivity of this assay is 0.004 mIU/L . The intra-assay CV and the inter-assay CV of the assay were 7.45% and 10.45%, respectively . The recovery rate was 91.4% -102.4% . The examination result of the assay correlates well with that of ACS-180 system . CONCLUSION: The CLELA for detecting TSH is low-cost, sensitive, specific and stable, and therefore may have a promising prospect in clinical application.

Anal Chem, 2004 Sep 15, 76(18), 5465 - 71
Pinched flow fractionation: continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel; Yamada M et al.; A concept of "pinched flow fractionation" for the continuous size separation and analysis of particles in microfabricated devices has been proposed and demonstrated . In this method, particles suspended in liquid were continuously introduced into a microchannel having a pinched segment and were aligned to one sidewall in the pinched segment by another liquid flow without particles . The particles were then separated perpendicularly to the flow direction according to their sizes by the spreading flow profile inside the microchannel . Polymer microbeads were successfully separated, and the effects of the flow rate and channel shapes on the separation performance were examined . Also, separated particles were collected independently by making branches at the end of the pinched segment . Since this method utilizes only the laminar flow profile inside a microchannel, complicated outer field control could be eliminated, which is usually required for other kinds of particle separation methods such as field flow fractionation . Also, this method can be applied both for particle size analysis and for preparation of monodispersed particles, since separation can be rapidly and continuously performed.

Ying Yong Sheng Tai Xue Bao, 2004 Jun, 15(6), 1000 - 4
{Effect of fertilization on soil enzymes and microbes in Larix gemlinii plantations}; Chen L; One year-old second generation stand and 14 year- and 34 year-old first generation stands in larch plantations were studied at the Maoershan Mt . Experimental Forest Farm . The forestland of these three stands was treated with various fertilizations . The results showed that fertilization could promote or restrain soil enzyme activity and microbial amount with different degrees, and its effect on soil physiological activity was more apparent . The effect of the same fertilization treatments on soil enzyme activity and microbial amount was different at different development stages of the stands . For one year-old stand, the best fertilization scheme was treatment 9, its soil enzyme activities (catalase, proteinase, polyphenoloxidase, urease, and saccharase), total microbial amount, and amount of bacteria, actinomyces and fungus being increased by 413.49%, 22.10%, 20.56%, 220.00%, 49.46%, 238.88%, 247.24%, 106.70%, and 366.67%, respectively, as compared with the control . For 34 year-old stand, the best fertilization scheme was treatment 5, its soil enzyme activities (catalase, proteinase, polyphenoloxidase, urease, and saccharase), total microbial amount, and amount of bacteria and fungus was increased by 30.44%, 16.91%, 0.22%, 43.06%, 124.18%, 119.92%, 87.66%, and 17.57%, respectively in rhizosphere soil, and by 24.55%, 77.01%, 168.62%, 251.85%, 183.33%, 250.0%, 38.24% and 128.57%, respectively in non-rhizosphere soil . For 14 year-old young stand, it needed a proper amount of nitrogen fertilizer and organic mineral fertilizers, and the better fertilization schemes were treatments 2 and 9 . The soil enzyme activities (catalase, proteinase, and urease) in treatment 2 was increased by 44.39%, 94.83%, and 4.62%, respectively in rhizosphere soil, and by 13.98%, 10.70% and 129.76%, respectively in non-rhizosphere soil . Total soil microbial amount and the amount of bacteria and fungus in treatment 9 was increased by 176.49%, 266.63%, and 198.04%, respectively in rhizosphere soil, and by 275.56%, 66.67% and 143.75%, respectively in non-rhizosphere soil.

Leuk Lymphoma, 2004 Jun, 45(6), 1167 - 73
Quantification of CD38 expression in B-cell chronic lymphocytic leukemia (B-CLL): a comparison between antibody binding capacity (ABC) and relative median fluorescence (RMF); Dignum HM et al.; We have previously shown that quantification of CD38 expression using microbeads of specific antibody binding capacity (ABC) improves the prognostic value of CD38 expression in B-cell chronic lymphocytic leukemia, particularly for Binet Stage A patients . Quantification of CD38 expression using beads is expensive, time consuming and could be difficult to implement in a routine clinical laboratory . The calculation of relative median fluorescence (RMF) using the median fluorescence intensities of the test and control samples, is even more simply and cheaply obtained by flow cytometry and could be used as an alternative way of quantifying antigen expression . The present study demonstrates that RMF is an effective prognostic indicator in B-CLL that correlates closely with ABC in predicting disease-specific survival and time to progression for all patients . RMF predicted overall survival and time to progression in all patients (P < 0.0001 for both), in Binet Stage A patients (P < 0.0001 for both) and in Stage A patients under 60 years (P = 0.0299 and P = 0.0143, respectively) . ABC predicted overall survival and time to progression in all patients (P < 0.0001 for both) in Stage A patients (P = 0.0024 and P < 0.0001, respectively) and in Stage A patients under 60 (P = 0.0379 and P = 0.0032, respectively) . RMF is more effective than percentage CD38 positivity > 30% or > 20% in predicting disease-specific survival in Stage A patients of all ages (CD38 < > 30%: P = 0.0853, CD38 < > 20%: P = 0.0894) and in those under 60 years old (CD38 < > 30%: P = 0.5438, CD38 < > 20%: P = 0.2872) . Also, RMF is more effective in predicting time to progression of Binet Stage A patients less than 60 years (P = 0.0143), while percentage CD38 positivity of 30%, 20% or 7% did not achieve statistical significance (P = 0.1103, = 0.0547, = 0.3399, respectively) . We suggest that CD38 RMF could be used clinically as an alternative to ABC to identify patients with B-CLL that are likely to progress and require early treatment.

J Proteome Res, 2004 Jul-Aug, 3(4), 736 - 42
Comparison of multiplexed techniques for detection of bacterial and viral proteins; Rao RS et al.; Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies . The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range . The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization . Both technologies were capable of sensitive multiplexed detection.

Hum Mol Genet, 2004 Oct 1, 13 Spec No 2, R187 - 94
Genetics of infectious diseases; Frodsham AJ et al.; Infectious diseases represent a major health problem worldwide, both in terms of morbidity and mortality . A complex combination of environmental, pathogen and host genetic factors plays a role in determining both susceptibility to particular microbes and the course of infection . Numerous studies have now mapped and identified relevant genes using a variety of both family-based and population-based approaches . Much interest has been focused on susceptibility to malaria, HIV/AIDS and mycobacterial infection, but other bacterial, viral and parasitic diseases are receiving increasing attention . Some major genes have been identified by genome scans of multi-case families, and mouse genetics has contributed to mapping and identification of a few genes . However, the great majority of known susceptibility loci emerged from screening of likely candidate genes . The emerging picture is of highly polygenic diseases, with occasional major genes, along with significant inter-population heterogeneity . This genetic architecture likely reflects the role that evolutionary selection has played in generating and maintaining a diverse repertoire of susceptibility/resistance loci, most with individually small effects . Genome-wide association studies with large sample sizes will be required to define the majority of the relevant polygenes.

Clin Infect Dis, 2004 Sep 1, 39(5), 702 - 9 Epub 2004 Aug 12.
Disinfection and sterilization in health care facilities: what clinicians need to know; Rutala WA et al.; All invasive procedures involve contact between a medical device or surgical instrument and a patient's sterile tissue or mucous membranes . A major risk of all such procedures is the introduction of pathogenic microbes that could lead to infection . Failure to properly disinfect or sterilize reusable medical equipment carries a risk associated with breach of the host barriers . The level of disinfection or sterilization is dependent on the intended use of the object: critical items (such as surgical instruments, which contact sterile tissue), semicritical items (such as endoscopes, which contact mucous membranes), and noncritical items (such as stethoscopes, which contact only intact skin) require sterilization, high-level disinfection, and low-level disinfection, respectively . Cleaning must always precede high-level disinfection and sterilization . Users must consider the advantages and disadvantages of specific methods when choosing a disinfection or sterilization process . Adherence to these recommendations should improve disinfection and sterilization practices in health care facilities, thereby reducing infections associated with contaminated patient-care items.

J Environ Qual, 2004 Sep-Oct, 33(5), 1595 - 605
Biological effects of wood ash application to forest and aquatic ecosystems; Aronsson KA et al.; The present review aims to summarize current knowledge in the topic of wood ash application to boreal forest and aquatic ecosystems, and the different effects derived from these actions . Much research has been conducted regarding the effects of wood ash application on forest growth . Present studies show that, generally speaking, forest growth can be increased on wood ash-ameliorated peatland rich in nitrogen . On mineral soils, however, no change or even decreased growth have been reported . The effects on ground vegetation are not very clear, as well as the effects on fungi, soil microbes, and soil-decomposing animals . The discrepancies between different studies are for the most part explained by abiotic factors such as variation in fertility among sites, different degrees of stabilization, and wood ash dosage used, and different time scales among different studies . The lack of knowledge in the field of aquatic ecosystems and their response to ash application is an important issue for future research . The few studies conducted have mainly considered changes in water chemistry . The biotoxic effects of ash application can roughly be divided into two categories: primary and secondary . Among the primary effects is toxicity deriving from compounds in the wood ash and cadmium is probably the worst among these . The secondary effects of wood ash are generally due to its alkaline capacity and a release of ions into the soil and soil water, and finally, watercourses and lakes . Given current knowledge, we would recommend site- and wood ash-specific application practices, rather than broad and general guidelines for wood ash application to forests.

Annu Rev Microbiol . 2004 May 14; {Epub ahead of print}
The Ecology and Genetics of Microbial Diversity; Kassen R et al.; Natural communities of microbes are often diverse, a fact that is difficult to reconcile with the action of natural selection in simple, uniform environments . We suggest that this apparent paradox may be resolved by considering the origin and fate of diversity in an explicitly ecological context . Here, we review insights into the ecological and genetic causes of diversity that stem from experiments with microbial populations evolving in the defined conditions of the laboratory environment . These studies highlight the importance of environmental structure in governing the fate of diversity and shed light on the genetic mechanisms generating diversity . We conclude by emphasizing the importance of placing detailed molecular-level studies within the context of a sound ecological and evolutionary framework . Expected online publication date for the Annual Review of Microbiology Volume 58 is September 8, 2004 . Please see for revised estimates.

Front Biosci, 2004 Sep 01, 9, 2177 - 82
Cell spreading controls balance of prestress by microtubules and extracellular matrix; Hu S et al.; The controversy surrounds the cellular tensegrity model . Some suggest that microtubules (MTs) must bear a significant portion of cell contractile stress (prestress) if tensegrity is a useful model . Previously we have shown that for highly spread airway smooth muscle cells (areas>2500 microm2) MTs balance a significant but small potion (average 14%) of the prestress . To further explore if controlling the degree of cell spreading could modulate the portion of the prestress balanced by MTs, we utilized a recent method by which tractions are quantified in cells that are constrained within micropatterned adhesive islands of defined sizes on the surface of flexible polyacrylamide gels containing fluorescent microbeads . The prediction is that if MTs balance a portion of the contractile stress, then, upon their disruption, the portion of the stress balanced by MTs would shift to the substrate, causing an increase in traction and strain energy . We first activated the cells maximally with histamine and then disrupted the MTs with colchicine . Histamine resulted in an increase in intracellular calcium whereas ensuing colchicine addition in the presence of histamine did not change intracellular calcium concentration, suggesting there was no additional net increase in contractile stress inside the cell . We found that following disruption of MTs the increase in traction and strain energy varied with the degree of cell spreading: as the cell projected areas increased from 500 micrometer 2 to about 1800 micrometer 2, the percent increase in tractions decreased from 80% to about a few percent and the percent increase in strain energy decreased from 200% to almost zero percent, indicating the portion of the prestress balanced by MTs decreased as the cells increased spreading . These findings demonstrate that complementary role of the extracellular matrix and the MTs in balancing the prestress is controlled by the degree of cell spreading.

Proteomics, 2004 Sep, 4(9), 2519 - 21
A human proteome project with a beginning and an end; Humphery-Smith I; Research activities centered on the ensemble of and individual human proteins have taken on numerous guises, some of which fall under the banner of what could be defined as a Human Proteome Project (HPP) . However, the latter has yet to take-on the apparent global focus of its predecessor, the Human Genome Project . The reasons for this are both financial and technical . The disparate properties afforded to each protein by a 20-letter code render a single unifying approach difficult to implement, while the current limit of analytical detection has yet to deliver an entire proteome for even the simplest of microbes . The situation is complicated further by the fact that low abundance proteins dominate within any living cell . Thus, enhancement of signal-to-noise ratio by affinity ligands becomes of paramount importance if whole-organism proteomics is to be realized . The generation of such ligands (molecules exhibiting desirable affinity and selectivity for target) could provide the necessary focus and a task list with a definable beginning and end . Such a finite task list is considered essential if an HPP might one day deliver global coverage on a scale seen currently for the total DNA sequence of some 200 living organisms.

Endeavour, 2004 Sep, 28(3), 109 - 13
Fighting the 'microbe of sporting mania': Australian science and Antarctic exploration in the early 20th century; Roberts P; The 'Heroic Age' of Antarctic exploration, which occurred during the first 15 years of the 20th century, captured headlines around the world . Australia was no exception, especially as Australian scientists played important roles in several expeditions . Through participation in the British Antarctic Expedition of 1907-1909, two Australian scientists - T.W . Edgeworth David (1858-1934) and Douglas Mawson (1882-1958) - became genuine national heroes, mainly through being members of the first party to reach the South Magnetic Pole area . At a superficial level, the vehicle of Antarctic exploration placed science at the forefront of public awareness, fulfilling David's ambition for greater recognition of science and scientists, especially considering the high level of public interest in sport . However, although David and Mawson gave Antarctic exploration a scientific veneer, simply through their status as scientists, the public viewed them as heroes because they had endured great hardships and conquered a point on the map in the name of science and the Empire.

Vet Immunol Immunopathol, 2004 Oct, 101(3-4), 171 - 8
Evaluation of elutriation and magnetic microbead purification of canine monocytes; de Carvalho CM et al.; An elutriation technique was developed to obtain large quantities of pure canine monocytes . Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient . Then, the PBMC were separated by an elutriation procedure . We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody . This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads . The cell viability was more than 95% and apoptotic cells were less than 10% . The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR) . A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+ . Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.

Water Res, 2004 Oct, 38(17), 3693 - 8
Plasticizer metabolites in the environment; Horn O et al.; Earlier work with pure cultures had shown that the interaction of microbes with plasticizers leads to the formation of metabolites including 2-ethylhexanoic acid and 2-ethylhexanol that resist further degradation . The presence of these metabolites is now reported in a variety of environmental samples . Thus, even in a complex ecosystem, when plasticizers are degraded, the breakdown is not complete and significant amounts of 2-ethylhexanoic acid and 2-ethylhexanol are observed . These compounds have been shown to exhibit acute toxicity using Microtox, Daphnia, rainbow trout and fathead minnow toxicity assays . Since it is already well established that plasticizers are ubiquitous in the environment, it is expected that their recalcitrant metabolites will also be ubiquitous . This is of concern because, while the plasticizers do not exhibit acute toxicity, their metabolites do.

Chemosphere, 2004 Nov, 57(6), 471 - 80
Adsorption-desorption, persistence and leaching behavior of thifluzamide in alluvial soil; Gupta S et al.; Investigations were undertaken to study the adsorption-desorption, persistence and leaching of thifluzamide (2',6'-dibromo-2-methyl-4'-trifluoromethoxy-4-trifluoro methyl-1,3-thiazole-5-carboxanilide) in an alluvial soil under laboratory conditions . The adsorption-desorption studies were carried out using batch equilibration technique . The results revealed high but weak adsorption of thifluzamide in alluvial soil with K(F) value of 9.62 and 'n' value of 0.63 . About 47-62% of the adsorbed amount got desorbed in four desorption cycles, which further substantiate the hypothesis of weak binding . The hysteresis coefficient varied from 0.19 to 0.40 . Persistence studies carried out at three concentration levels (0.1, 1.0 and 10.0 microgg(-1)) and under three moisture conditions (air-dry, field capacity moisture and submerged) revealed that thifluzamide is a persistent chemical and only 19.5-54.0% dissipation was recorded on 90th day . However, it appears that aerobic microbes are more efficient in degrading thifluzamide than anaerobic microbes . The preliminary leaching studies carried out in the laboratory revealed that thifluzamide was moderately mobile in alluvial soil . Only small amounts (<1%) were recovered from leachate fractions whereas major portion remained in 0-15 cm soil depth.

J Mater Sci Mater Med, 1998, 9(2), 109 - 15
X-ray diffraction of bone at the interface with hydroxyapatite-coated versus uncoated metal implants; Savarino L et al.; The microstructural characteristics of the newly formed bone tissue at the interface with hydroxyapatite-coated and uncoated stainless steel pins used in an external fracture fixation system have been evaluated . The bone far from the interface was used as a control . Pins were transversally inserted into the diaphyses of sheep tibiae and were loaded in for six weeks . Three sheep received coated pins and two received uncoated pins . Crystallographic habit and mineralization of the implant-facing bone were evaluated . Moreover, lattice parameters of bone apatite were measured and hydroxyapatite (HA) coating degradation was investigated, by means of conventional and microbeam X-ray diffraction (XRD) . In coated pins, six weeks after the implantation the newly formed bone tissue at the interface did not reach complete maturation, but the presence of the implant did not alter the apatite lattice structure; the lattice parameters did not show statistically significant variations with respect to those observed in the control bone . In uncoated pins, bone tissue rarely appeared totally mineralized and lattice parameters were significantly different with respect to those observed in the bone far from the implant . HA particles were observed spreading in the bone-facing coated pins; the XRD pattern of bone apatite surrounding HA particles was unmodified . It was concluded that HA coatings improved the bone remodelling process during pin fixation in comparison to uncoated pins and did not alter the crystallographic habit of apatite .

J Mater Sci Mater Med, 2001 Aug, 12(8), 679 - 82
Porous HA ceramic for bone replacement: Role of the pores and interconnections - experimental study in the rabbit; Flautre B et al.; Hydroxyapatite (HA) porous ceramics are increasingly used in biomedical applications . Their physical characteristics, such as porous volume, require perfect control of the pore shape, as well as the number and the size of their interconnections . The aim of our study was to evaluate a new HA ceramic using polymethylmethacrylate microbeads (PMMA) as the porous agent . Four interconnection sizes (30, 60, 100 and 130 microm) with a 175-260 microm pore size and three pore sizes (175-260, 260-350 and 350-435 microm) for a 130 microm interconnection size were tested . Various HA implants were appraised by microscopic evaluation in a 4.6x10 mm rabbit femur cancellous bone defect 12 weeks after implantation . The best osteoconduction result was obtained in the center of the ceramic by means of a 130 microm interconnection size and a 175-260 microm mean pore size . Bone formation obtained within the pores was double that obtained in our previous study where naphtalen microbeads were used as the porous agents .

J Mater Sci Mater Med, 1999 Dec, 10(12), 811 - 814
Histological aspects in bone regeneration of an association with porous hydroxyapatite and bone marrow cells; Flautre B et al.; The osteogenic potential of an association of two kinds of hydroxyapatite (HA) porous ceramics with autologous bone marrow cells cultured with or without dexamethasone (10^-8M) addition in the culture medium and non-cultured rabbit marrow stromal stem cells (MSCs) was tested after 4 weeks of implantation in the dorsal muscles of spine in rabbit . A significantly higher number of rabbits with implants containing bone tissue inside pores were obtained with 10^7 cells ml^-1 cultured treated with Dex . In the HA porous ceramic using naphtalen as porogen agent, the bone recolonization remains only at the periphery of implants and in the second row of pores, while in the HA porous ceramic using polymethacrylate (PMMA) microbeads as porogen agent, the bone recolonization is observed in the depth of implants . In the PMMA HA group, the Kruskal-Wallis variance analysis between the rabbits is significantly different with the percentage of number of occupied pores and occupied pores with bone tissue is different (p\,\lt\,0.05) .

Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13495 - 500 Epub 2004 Sep 02.
Targeted cytoplasmic irradiation induces bystander responses; Shao C et al.; The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure . Much of this questions the current dogma of direct DNA damage driving response in irradiated systems . In this study, we have used a charged-particle microbeam to target individual helium ions ((3)He(2+)) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts . We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one (3)He(2+) ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population . Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted . The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively . By using the probe 4-amino-5-methylamino-2',7'-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin . This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

J Immunol Methods, 2004 Aug, 291(1-2), 79 - 91
Responses of peptide-specific T cells to stimulation with polystyrene beads carrying HLA class I molecules loaded with single peptides; Chersi A et al.; Cell-sized microbeads carrying single peptide-loaded HLA class I molecules were prepared for HLA-A2 and HLA-B7 by a simple procedure which transfers single peptide-loaded HLA class I molecules from cultured cells to polystyrene beads using anti-peptide antibodies directed to an intracellular segment of HLA-A alpha chains . The surface density of peptide-loaded HLA class I molecules on beads was comparable to that on the peptide-loaded cells . HLA-A2 beads loaded with an HCV peptide HCV1073 were tested for stimulation activity on an HCV1073-specific CD8+ T cell clone NS3-1 . A substantial level of gamma-IFN production was induced . The stimulation was peptide-specific . The efficiency was dependent on the bead concentration and the surface HLA class I density on beads and enhanced significantly by co-coupling of anti-CD28 to peptide-loaded beads . The peptide-loading efficiency on HLA class I molecules and the transfer efficiency of HLA class I molecules to polystyrene beads were reasonably high for HLA-A2 and HLA-B7 . Thus, polystyrene beads carrying these single peptide-loaded HLA class I molecules are potentially useful in further analysis of the co-stimulatory or inhibitory factors involved in CD8+ T cell responses and eventually in detection of cytotoxic T cells in PBLs.

J Immunol Methods, 2004 Aug, 291(1-2), 1 - 10
The methodology for determining the efficacy of antibody-mediated immunity; Casadevall A; The basic method for evaluating the efficacy of antibody-mediated immunity (AMI) dates from the 1890s and involves the administration of specific Ab to an immunologically naive host followed by microbial or toxin challenge . Other methods used to evaluate AMI involve correlating the presence of specific Ab with resistance to microbial disease and associating susceptibility to certain microbes with host immunoglobulin deficits . Unfortunately, each method has theoretical and practical problems that limit their usefulness when negative results are obtained . The application of hybridoma technology to investigate the efficacy of AMI has shown that it is possible to generate protective monoclonal antibodies even to microbes for which the standard methodologies indicate no role for AMI . Furthermore, studies with monoclonal antibodies suggest various explanations for the inability of standard methods to demonstrate the potential efficacy of AMI for certain pathogens.

Cancer Res, 2004 Sep 1, 64(17), 5963 - 72
Molecular features of the transition from prostatic intraepithelial neoplasia (PIN) to prostate cancer: genome-wide gene-expression profiles of prostate cancers and PINs; Ashida S et al.; To characterize the molecular feature in prostate carcinogenesis and the putative transition from prostatic intraepithelial neoplasia (PIN) to invasive prostate cancer (PC), we analyzed gene-expression profiles of 20 PCs and 10 high-grade PINs with a cDNA microarray representing 23,040 genes . Considering the histological heterogeneity of PCs and the minimal nature of PIN lesions, we applied laser microbeam microdissection to purify populations of PC and PIN cells, and then compared their expression profiles with those of corresponding normal prostatic epithelium also purified by laser microbeam microdissection . A hierarchical clustering analysis separated the PC group from the PIN group, except for three tumors that were morphologically defined as one very-high-grade PIN and two low-grade PCs, suggesting that PINs and PCs share some molecular features and supporting the hypothesis of PIN-to-PC transition . On the basis of this hypothesis, we identified 21 up-regulated genes and 63 down-regulated genes commonly in PINs and PCs compared with normal epithelium, which were considered to be involved in the presumably early stage of prostatic carcinogenesis . They included AMACR, OR51E2, RODH, and SMS . Furthermore, we identified 41 up-regulated genes and 98 down-regulated genes in the transition from PINs to PCs; those altered genes, such as POV1, CDKN2C, EPHA4, APOD, FASN, ITGB2, LAMB2, PLAU, and TIMP1, included elements that are likely to be involved in cell adhesion or the motility of invasive PC cells . The down-regulation of EPHA4 by small interfering RNA in PC cells lead to attenuation of PC cell viability . These data provide clues to the molecular mechanisms underlying prostatic carcinogenesis, and suggest candidate genes the products of which might serve as molecular targets for the prevention and treatment of PC.

Curr Opin Immunol, 2004 Oct, 16(5), 538 - 44
Toll-like receptors and other links between innate and acquired alloimmunity; Goldstein DR; Innate immunity represents the first line of defense against invading pathogens and noxious stimuli . The Toll-like receptors (TLRs) are essential innate immune receptors that alert the immune system to the presence of invading microbes . Emerging evidence shows that TLR signaling is important in allograft rejection . In a murine model, the rejection of minor mismatched allografts cannot occur in the absence of MyD88, an important TLR signal adaptor protein, owing to a defect in dendritic cell maturation, which leads to diminished T-helper cell type 1 immune responses . A recent clinical study also suggests that recipients with a mutant TLR4 genotype manifest reduced lung allograft rejection . Thus, innate immune signaling via TLRs is important for alloimmunity.

Nat Biotechnol, 2004 Sep, 22(9), 1105 - 9
Problems in monitoring horizontal gene transfer in field trials of transgenic plants; Heinemann JA et al.; Transgenic crops are approved for release in some countries, while many more countries are wrestling with the issue of how to conduct risk assessments . Controls on field trials often include monitoring of horizontal gene transfer (HGT) from crops to surrounding soil microorganisms . Our analysis of antibiotic-resistant bacteria and of the sensitivity of current techniques for monitoring HGT from transgenic plants to soil microorganisms has two major implications for field trial assessments of transgenic crops: first, HGT from transgenic plants to microbes could still have an environmental impact at a frequency approximately a trillion times lower than the current risk assessment literature estimates the frequency to be; and second, current methods of environmental sampling to capture genes or traits in a recombinant are too insensitive for monitoring evolution by HGT . A model for HGT involving iterative short-patch events explains how HGT can occur at high frequencies but be detected at extremely low frequencies.

Oecologia, 2004 Oct, 141(2), 254 - 68 Epub 2004 Aug 27.
Precipitation pulses and carbon fluxes in semiarid and arid ecosystems; Huxman TE et al.; In the arid and semiarid regions of North America, discrete precipitation pulses are important triggers for biological activity . The timing and magnitude of these pulses may differentially affect the activity of plants and microbes, combining to influence the C balance of desert ecosystems . Here, we evaluate how a "pulse" of water influences physiological activity in plants, soils and ecosystems, and how characteristics, such as precipitation pulse size and frequency are important controllers of biological and physical processes in arid land ecosystems . We show that pulse size regulates C balance by determining the temporal duration of activity for different components of the biota . Microbial respiration responds to very small events, but the relationship between pulse size and duration of activity likely saturates at moderate event sizes . Photosynthetic activity of vascular plants generally increases following relatively larger pulses or a series of small pulses . In this case, the duration of physiological activity is an increasing function of pulse size up to events that are infrequent in these hydroclimatological regions . This differential responsiveness of photosynthesis and respiration results in arid ecosystems acting as immediate C sources to the atmosphere following rainfall, with subsequent periods of C accumulation should pulse size be sufficient to initiate vascular plant activity . Using the average pulse size distributions in the North American deserts, a simple modeling exercise shows that net ecosystem exchange of CO2 is sensitive to changes in the event size distribution representative of wet and dry years . An important regulator of the pulse response is initial soil and canopy conditions and the physical structuring of bare soil and beneath canopy patches on the landscape . Initial condition influences responses to pulses of varying magnitude, while bare soil/beneath canopy patches interact to introduce nonlinearity in the relationship between pulse size and soil water response . Building on this conceptual framework and developing a greater understanding of the complexities of these eco-hydrologic systems may enhance our ability to describe the ecology of desert ecosystems and their sensitivity to global change.

Ying Yong Sheng Tai Xue Bao, 2004 Apr, 15(4), 707 - 11
{Chemical mechanism of exotic weed invasion}; Wang P et al.; The ecological and economic impacts of exotic invasive weeds have been followed with interest . A successful invasion of exotic weeds depends not only on their bio-ecological traits, but also on some specific mechanisms of invasion . The clarification of the invasive mechanism of exotic weeds will be beneficial to predict and manage on them . In the process of establishment, reproduction and spreading, an exotic species has to become dominant in the interactions with native ones at new habitats, and then invades successfully . Among invasive mechanisms, the chemical interaction between exotic and native weeds should not be neglected . Previous studies showed that allelopathy plays an important role in exotic weed invasion . In fact, plants are able to defend or inhibit animals, plants and microbes from new habitats by using some specific chemicals . The chemical mechanism of exotic weed invasion might involve each aspect of plant chemical ecology . Therefore, chemical ecological characteristics of exotic weeds should be regarded as one of the most significant factors on predicting invasive species . The studies on chemical mechanism of exotic weed invasion will be one of the important aspects in future.

Radiat Res, 2004 Sep, 162(3), 326 - 31
Comparison of microdosimetric simulations using PENELOPE and PITS for a 25 keV electron microbeam in water; Mainardi E et al.; The calculations presented compared the performances of two Monte Carlo codes used for the estimation of microdosimetric quantities: Positive Ion Track Structure code (PITS) and a main user code based on the PENetration and Energy Loss of Positrons and Electrons code (PENELOPE-2000) . Event-by-event track structure codes like PITS are believed to be superior for microdosimetric applications, and they are written for this purpose . PITS tracks electrons in water down to 10 eV . PENELOPE is one of the few general-purpose codes that can simulate random electron-photon showers in any material for energies from 100 eV to 1 GeV . The model used in the comparison is a water cylinder with an internal scoring geometry made of spheres 1 microm in diameter where the scoring quantities are calculated . The source is a 25 keV electron pencil beam impinging normally on the sphere surface . This work shows only the lineal energy y and spectra graphical presentation as a function of y since for microdosimetry and biology applications, and for discussion of radiation quality in general, these results are more appropriate . The computed PENELOPE results are in agreement with those obtained with the PITS code and published previously in this journal . This paper demonstrates PENELOPE's usefulness at low energies and for small geometries . What is still needed are experimental results to confirm these analyses.

Indoor Air, 2004 Oct, 14(5), 351 - 9
Personal exposures to particles and microbes in relation to microenvironmental concentrations; Toivola M et al.; The aim of this study was to compare the personal exposures of particles and microbes with the exposure being assessed by stationary samplers in the main microenvironments, i.e . home and the workplace . A random sample of 81 elementary school teachers in eastern Finland were selected to perform the two wintertime 24-h measurement periods . Particle mass concentration, black smoke (BS) concentration and concentrations of viable and total microorganisms on the sampled filters were determined using personal exposure sampling and microenvironmental measurements in homes and workplaces . In this paper, the correlations between different pollutants in each environment and correlations between personal exposures and home and work concentrations are presented . The results show that personal BS exposures correlated with both home and work BS concentrations . Furthermore, the concentrations of viable fungi and bacteria were related between personal and home concentrations . The time weighted microenvironmental model underestimated the personal exposures of particle mass, viable fungi, total fungi and total bacteria concentrations but the model might satisfactorily assess personal exposure to concentrations of BS and viable bacteria . The mass concentration of total fungi and bacteria was <1% of the total particle mass concentration . PRACTICAL IMPLICATIONS: Stationary samples are only surrogate measures of personal exposures . Personal exposure measurements conducted on individuals' breathing zone are needed to assess the exposure to particles and microbes . The time weighted microenvironmental model is a useful method to assess personal exposure to combustion related particles and viable bacteria concentrations but the model underestimates personal exposures of particle mass, viable fungi, total fungi and total bacteria concentrations.

Org Lett, 2004 Sep 2, 6(18), 3163 - 5
Biotransformation of quinovic acid glycosides by microbes: direct conversion of the ursane to the oleanane triterpene skeleton by Nocardia sp . NRRL 5646; Cheng ZH et al.; {reaction: see text} Quinovic acid glycosides were microbially deglycosylated by a Nocardia sp . to their aglycon quinovic acid and its biogenetic counterpart, cincholic acid (3), via an unprecedented carbon skeleton rearrangement involving a methyl group migration . The structures of the metabolites were established by ESI-LC/MS and 2D-NMR techniques.

Med Hypotheses, 2004, 63(4), 560 - 6
Ice as a reservoir for pathogenic human viruses: specifically, caliciviruses, influenza viruses, and enteroviruses; Smith AW et al.; Hundreds of isolates of viable bacteria and fungi have been recovered from ancient ice and permafrost . Evidence supports the hypothesis that viral pathogens also are preserved in ice repositories, such as glaciers, ice sheets, and lake ice . Proof may depend upon narrowing the search by applying specific criteria, which would target candidate viruses . Such criteria include viral pathogens likely to occur in great abundance, likely to be readily transported into ice, and then participate in ongoing disease cycles suggestive of their having been deposited in and subsequently released from ice . Caliciviruses, influenza A, and some enteroviruses appear to satisfy all three criteria . Environmental ice appears to be an important abiotic reservoir for pathogenic microbes . World health and eradication of specific pathogens could be affected by this huge reservoir .

Oncogene, 2004 Aug 23, 23(38), 6524 - 34
The epidemiology of non-Hodgkin's lymphoma; Fisher SG et al.; The incidence of non-Hodgkin's lymphoma (NHL) has doubled over the past two decades in the US and most other westernized countries . While improved cancer reporting, changes in lymphoma classification, and increases in AIDS-associated lymphomas have contributed to the startling escalation of disease incidence, these factors are estimated to account for only about 50% of the increase in observed incidence . The elucidation of etiologic factors and their mechanistic role in the pathogenesis of this malignancy are critical to advancements in disease prevention and treatment . Current evidence suggests that factors/conditions that precipitate either chronic antigenic stimulation or immunosuppression may provide a preferential milieu for development of NHL . High rates of lymphoma have been observed among individuals with autoimmune disease, organ transplants, and primary or acquired immunodeficiencies . Ultraviolet radiation, previously demonstrated to have an immunosuppressive effect, has also been suggested as a possible risk factor for NHL . Several pathogens have been linked to the risk of lymphoma, including Epstein-Barr virus, human immunodeficiency virus, human T-cell lymphotropic virus-1, Helicobacter pylori, hepatitis C, and simian virus 40 . Whether these microbes are responsible for specific genetic mutations that initiate tumor growth, antigenic stimulation leading to B-cell proliferation, and increased potential of random cell replication errors, or immunosuppression, which thereby promotes tumor growth, has not been clearly delineated . Other exogenous factors which have been implicated in lymphomagenesis are chemicals and agricultural exposures, hair dyes, and blood transfusions . We must build on our current knowledge regarding the etiology of NHL in order that prevention, treatment, and ultimately, cure of this malignancy becomes a reality.

Oncogene, 2004 Aug 23, 23(38), 6341 - 8
Infections and immune factors in cancer: the role of epidemiology; Kinlen L; An infective, mostly viral, basis has been found in an increasing number of different human cancers . In all cases, the neoplasm is a rare response to the relevant infection, which is usually present in persistent form, and requiring specific cofactors for malignancy to develop . In some cases, epidemiological evidence of infectivity preceded and promoted identification of the specific infection involved and even the discovery of the microbe itself, as in Burkitt's lymphoma and cervix cancer . In other cases, the discovery of the agent came first as in stomach and nasopharynx cancers, and epidemiology has been concerned mainly with confirming the relationship, measuring the size of the risk and identifying cofactors . Infection-linked cancers include some of the commonest malignancies in certain large world regions, amounting to over 20% of all cancer in the developing countries . In addition to these cancers are others such as childhood leukaemia that show features indicative of an infective basis though no underlying agent has been identified . Advances in this field invite speculation about possible future discoveries and how these might be promoted . However, in that majority of cancers that are unrelated to sexual behaviour, there will be nothing even at the population level to suggest an infective basis because what is transmitted from one individual to another is not the neoplasm itself, but the underlying, often silent, infection to which the malignancy is an uncommon response . The increasing prevalence of immune impairment in human populations, as a result of the use of immunosuppressive drugs with organ transplants and the spread of HIV infection, has produced marked effects on cancer incidence in the affected groups including increases, of skin cancers, non-Hodgkin's lymphoma and Kaposi's sarcoma and to a lesser extent of many other cancers, in some cases at least due to the release from immunological control of incipient infection-based malignancies.

J Immunol, 2004 Sep 1, 173(5), 3020 - 6
Bcl-2 transduction protects human endothelial cell synthetic microvessel grafts from allogeneic T cells in vivo; Zheng L et al.; T cell interactions with vascular endothelial cells (EC) are of central importance for immune surveillance of microbes and for pathological processes such as atherosclerosis, allograft rejection, and vasculitis . Animal (especially rodent) models incompletely predict human immune responses, in particular with regard to the immunological functions of EC, and in vitro models may not accurately reflect in vivo findings . In this study, we describe the development of an immunodeficient SCID/bg murine model combining a transplanted human synthetic microvascular bed with adoptive transfer of human T lymphocytes allogeneic to the cells of the graft that more fully recapitulates T cell responses in natural tissues . Using this model, we demonstrate that transduced Bcl-2 protein in the engrafted EC effectively prevents injury even as it enhances T cell graft infiltration and replication.

Ying Yong Sheng Tai Xue Bao, 2004 May, 15(5), 899 - 904
{Methods for studying soil microbial diversity}; Zhong W et al.; This paper gave a review on the main methods for studying soil microbial diversity . Traditionally, the analysis of soil microbial communities relied on culturing techniques, using a variety of culture media . However, only a small fraction of the soil microbial community has been cultured and isolated with this approach . Other methods such as Biolog GN analysis, phospholipids fatty acids analysis and nucleic acid-based analysis can be used to study and characterize soil microbes which currently cannot be cultured, and to get more and complete information about soil microbial community.

Ying Yong Sheng Tai Xue Bao, 2004 May, 15(5), 867 - 70
{Effect of organic materials in controlling cucumber seedling diseases}; Yuan F et al.; In this work, pot experiments with organic materials were carried out to study the alleviation of Fusarim wilt and Rhizictonia wilt of cucumber and the changes of soil microorganism . The results showed that rice straw, pig feces and wood chip could alleviate Fusarium wiltand Rhizoctonia wilt of cucumber, and the effect of rice strawwas most significant, followed by pig feces and wood chip . The amount of soil microbes was higher in organic materials treatments than in control . The total amount of actinomyces and epidhyte which can inhibit the growth of pathogen was the highest in rice straw treatment, followed by pig feces treatment . No significant change of soil microbial amount was found in wood chip treatment.

Plant Cell, 2004 Sep, 16(9), 2514 - 28 Epub 2004 Aug 19.
Interaction-dependent gene expression in Mla-specified response to barley powdery mildew; Caldo RA et al.; Plant recognition of pathogen-derived molecules influences attack and counterattack strategies that affect the outcome of host-microbe interactions . To ascertain the global framework of host gene expression during biotrophic pathogen invasion, we analyzed in parallel the mRNA abundance of 22,792 host genes throughout 36 (genotype x pathogen x time) interactions between barley (Hordeum vulgare) and Blumeria graminis f . sp hordei (Bgh), the causal agent of powdery mildew disease . A split-split-plot design was used to investigate near-isogenic barley lines with introgressed Mla6, Mla13, and Mla1 coiled-coil, nucleotide binding site, Leu-rich repeat resistance alleles challenged with Bgh isolates 5874 (AvrMla6 and AvrMla1) and K1 (AvrMla13 and AvrMla1) . A linear mixed model analysis was employed to identify genes with significant differential expression (P value < 0.0001) in incompatible and compatible barley-Bgh interactions across six time points after pathogen challenge . Twenty-two host genes, of which five were of unknown function, exhibited highly similar patterns of upregulation among all incompatible and compatible interactions up to 16 h after inoculation (hai), coinciding with germination of Bgh conidiospores and formation of appressoria . By contrast, significant divergent expression was observed from 16 to 32 hai, during membrane-to-membrane contact between fungal haustoria and host epidermal cells, with notable suppression of most transcripts identified as differentially expressed in compatible interactions . These findings provide a link between the recognition of general and specific pathogen-associated molecules in gene-for-gene specified resistance and support the hypothesis that host-specific resistance evolved from the recognition and prevention of the pathogen's suppression of plant basal defense.

Endocrinology, 2004 Dec, 145(12), 5832 - 9 Epub 2004 Aug 19.
Rapid, efficient isolation of murine gonadotropes and their use in revealing control of follicle-stimulating hormone by paracrine pituitary factors; Wu JC et al.; FSH and LH are produced only in gonadotropes, which are reported to comprise 3-12% of mammalian pituitaries . Factors made within the pituitary are powerful regulators of FSH and also influence LH expression, but their identities and cellular origins are unknown because it is impossible to isolate and individually analyze different pituitary cell types . In this study FSH-producing gonadotropes were specifically tagged in vivo with a transgenic cell surface antigen (H-2Kk) so they could be purified in vitro using paramagnetic anti-H-2Kk microbeads . After enzymatic dispersion of pituitary cells, it took 1 h or less to extract 55 +/- 5% of FSH-producing gonadotropes at 95 +/- 0.5% purity, as judged by immunostaining for FSH or prolactin . Although this procedure selected for FSH expression, the isolated gonadotropes were also enriched 22-fold for LH-containing cells . For studies aimed at understanding factors that control FSH transcription, the purified gonadotropes were treated with activin A, which increased FSH expression 480% above basal levels (d 3 of culture) . Coincubation of purified gonadotropes with pituitary nongonadotropes increased FSH expression 800% (d 3 of culture) . Follistatin, an activin-binding protein, decreased FSH expression 35-50%, suggesting that gonadotropes make some activin and/or other follistatin-sensitive molecule(s) that induce FSH . These data show that paracrine factors from pituitary nongonadotropes can play a major role in controlling FSHbeta at the pituitary level . The study presented here describes a rapid, reliable, and efficient method for isolating any specialized cell type, including all cells that produce endocrine hormones.

Oral Dis, 2004 Sep, 10(5), 298 - 305
Intracellular localization of Porphyromonas gingivalis thiol proteinase in periodontal tissues of chronic periodontitis patients; Rautemaa R et al.; OBJECTIVES: Porphyromonas gingivalis is a significant periodontal pathogen that has been shown in vitro to be able to invade gingival epithelial cells and grow intracellularly . The aim of the present study was to detect P . gingivalis in gingival tissues from chronic periodontitis (CP) patients . MATERIALS AND METHODS: Monoclonal antibodies specific to a cell membrane-bound thiol proteinase of P . gingivalis were used to detect the microbe in gingival tissues of CP patients (n = 13) by immunohistochemistry . The presence of P . gingivalis was also analysed by polymerase chain reaction (PCR) . RESULTS: Immunohistochemical analysis of the periodontal tissues revealed positive staining for P . gingivalis thiol proteinase in 11 of the 13 patients . Positive staining was mainly located intracellularly in the perinuclear region of the cytoplasm in the periodontal epithelial cells and it could be detected throughout the whole depth of both pocket and oral epithelium . The sensitivity of immunohistochemistry was found to be comparable with that of PCR . CONCLUSIONS: Our results provide in vivo evidence of the ability of P . gingivalis to enter human gingival epithelial cells . Intracellular localization of P . gingivalis contributes to its evasion of the host immune surveillance and eventually increases its resistance to conventional treatments of periodontal diseases.

J Evol Biol, 2004 Sep, 17(5), 1058 - 72
Diminishing returns in social evolution: the not-so-tragic commons; Foster KR; A challenge for evolutionary theory is to understand how cooperation can occur in the presence of competition and cheating, a problem known as the tragedy of commons . Here I examine how varying the fitness returns from reproductive competition or cooperation affects the negative impact of competition on a social group . Varying linear returns does not affect the impact of competition . However, diminishing returns, where additional investments in either competition or cooperation give smaller and smaller rewards, reduce the effects of competition on the group . I show that diminishing returns are common in many systems, including social vertebrates, microbes, social insects and mutualisms among species . This suggests that the tragedy of the commons is not so tragic and that the disruptive effects of competition upon social life will often be minor.

J Synchrotron Radiat, 2004 Sep 1, 11(Pt 5), 428 - 31 Epub 2004 Aug 17.
Source identification of PM10, collected at a heavy-traffic roadside, by analyzing individual particles using synchrotron radiation; Yue W et al.; Synchrotron radiation microbeam X-ray fluorescence (micro-SXRF) was used to analyze individual aerosol particles collected at a height of 2 m above a heavy-traffic roadside in a heavy-industrial area of Shanghai . A pattern recognition technique, which took micro-SXRF spectra of single aerosol particles as its fingerprint, was used to identify the origins of the particles . The particles collected from the environmental monitoring site are mainly from metallurgic industry (26%), unleaded gasoline automobile exhaust (15%), coal combustion (10%), cement dust (10%) and motorcycle exhaust (8%).

Appl Radiat Isot, 2004 Nov, 61(5), 771 - 4
Microdistributions of prospective BNCT-compound CuTCPH in tissue sections with a heavy ion microbeam; Stoliar P et al.; Microdistributions of the prospective BNCT-compound CuTCPH, a carborane-containing tetraphenylporphyrin with one Cu atom in its molecular structure, have been obtained in tissue sections of different organs of tumor-bearing and normal Syrian hamsters injected with the boron compound by employing a heavy ion microbeam . High resolution X-ray spectroscopy following micro-PIXE (Particle Induced X-ray Emission with micrometer-sized beams) with a focused (16)O ion beam was used . Focusing was performed with a heavy-ion scanning high-precision magnetic quadrupole triplet microprobe . Squamous Cell Carcinomas were induced on the right Cheek Pouch of Syrian Hamsters (HCP), sampled, cryo-sectioned and freeze-dried . Two-dimensional maps of elemental concentration were obtained by scanning the beam over the samples . Very non-uniform Cu concentrations were found in all sections.

Eur J Immunol, 2004 Sep, 34(9), 2620 - 9
Complement inhibitor membrane cofactor protein (MCP; CD46) is constitutively shed from cancer cell membranes in vesicles and converted by a metalloproteinase to a functionally active soluble form; Hakulinen J et al.; Human cell-surface protein CD46 protects cells from complement damage, regulates immune functions through signaling and acts as a receptor for certain pathogenic microbes . Multiple molecular weight isoforms of membrane bound CD46 are produced by alternative splicing of the CD46 mRNA in an area coding for the serine/threonine/proline-rich region or for the cytoplasmic tail . We demonstrate that CD46 becomes proteolytically modified on cell membranes . We observed that tumor cells liberated intact 60-65 kDa forms of CD46 into the cell culture medium on the surface of vesicles with a diameter of 200 nm . Furthermore, soluble CD46 (55-60 kDa) containing the glycosylated STP-region but lacking the hydrophobic transmembrane sequence and cytoplasmic domains was released from tumor cell membranes . The use of selective inhibitors indicated that CD46 release is due to specific cleavage by a metalloproteinase . Exposure of the cells to hydrogen peroxide (H2O2) or their detachment from the pericellular matrix increased the shedding of soluble CD46 . Both vesicular and soluble forms of CD46 remained functional and promoted C3b cleavage by factor I . The results show that the functional activity of CD46 is not restricted to the tumor cell membranes but can be liberated in vesicles and by a metalloproteinase .

Nature, 2004 Aug 12, 430(7001), 761 - 4
Bulk glasses and ultrahard nanoceramics based on alumina and rare-earth oxides; Rosenflanz A et al.; Although often regarded as a network-former in conventional silicate glasses, Al(2)O(3) alone cannot be obtained as a bulk glass . Until now, glasses comprising continuously linked {AlO(x)} polyhedra have been prepared in only a few systems under very fast cooling conditions, which limits their dimensions to a few millimetres . Yet it is desirable to prepare bulk, or monolithic, alumina-rich glasses, with the prospect of superior mechanical, chemical and optical properties . Here we report a novel process for preparing very-high-alumina glasses and nanoscale glass-ceramics . Fully dense bulk articles in net shape are obtained through viscous sintering of glass microbeads . Additional heat treatment of the consolidated glasses leads to fully crystallized transparent glass-converted nanoceramics with a hardness similar to that of alumina . This method avoids the impracticably high applied pressures (more than 1 GPa) that have been required in most cases to prepare nanocrystalline ceramics by sintering, owing to the concurrent nature of densification and grain growth under pressureless conditions . The reported techniques can be extended to form glasses and nanoceramics in other oxide systems that do not include a conventional glass-forming component.

Philos Trans R Soc Lond B Biol Sci, 2004 Jul 29, 359(1447), 1049 - 58
Environmental and social influences on emerging infectious diseases: past, present and future; McMichael AJ; During the processes of human population dispersal around the world over the past 50 000-100 000 years, along with associated cultural evolution and inter-population contact and conflict, there have been several major transitions in the relationships of Homo sapiens with the natural world, animate and inanimate . Each of these transitions has resulted in the emergence of new or unfamiliar infectious diseases.The three great historical transitions since the initial advent of agriculture and livestock herding, from ca . 10 000 years ago, occurred when: (i) early agrarian-based settlements enabled sylvatic enzootic microbes to make contact with Homo sapiens; (ii) early Eurasian civilizations (such as the Greek and Roman empires, China and south Asia) came into military and commercial contact, ca . 3000-2000 years ago, swapping their dominant infections; and (iii) European expansionism, over the past five centuries, caused the transoceanic spread of often lethal infectious diseases . This latter transition is best known in relation to the conquest of the Americas by Spanish conquistadores, when the inadvertent spread of measles, smallpox and influenza devastated the Amerindian populations.Today, we are living through the fourth of these great transitional periods . The contemporary spread and increased lability of various infectious diseases, new and old, reflect the combined and increasingly widespread impacts of demographic, environmental, behavioural, technological and other rapid changes in human ecology . Modern clinical medicine has, via blood transfusion, organ transplantation, and the use of hypodermic syringes, created new opportunities for microbes . These have contributed to the rising iatrogenic problems of hepatitis C, HIV/AIDS and several other viral infections . Meanwhile, the injudicious use of antibiotics has been a rare instance of human action actually increasing 'biodiversity'.Another aspect of this fourth transition is that modern hyper-hygienic living restricts microbial exposure in early life . This, in the 1950s, may have contributed to an epidemic of more serious, disabling, poliomyelitis, affecting older children than those affected in earlier, more endemic decades . As with previous human-microbe transitions, a new equilibrial state may lie ahead . However, it certainly will not entail a world free of infectious diseases . Any mature, sustainable, human ecology must come to terms with both the need for, and the needs of, the microbial species that help to make up the interdependent system of life on Earth . Humans and microbes are not "at war"; rather, both parties are engaged in amoral, self-interested, coevolutionary struggle . We need to understand better, and therefore anticipate, the dynamics of that process.

Proc R Soc Lond B Biol Sci, 2004 Jun 22, 271(1545), 1251 - 62
Only six kingdoms of life; Cavalier-Smith T; There are many more phyla of microbes than of macro-organisms, but microbial biodiversity is poorly understood because most microbes are uncultured . Phylogenetic analysis of rDNA sequences cloned after PCR amplification of DNA extracted directly from environmental samples is a powerful way of exploring our degree of ignorance of major groups . As there are only five eukaryotic kingdoms, two claims using such methods for numerous novel 'kingdom-level' lineages among anaerobic eukaryotes would be remarkable, if true . By reanalysing those data with 167 known species (not merely 8-37), I identified relatives for all 8-10 'mysterious' lineages . All probably belong to one of five already recognized phyla (Amoebozoa, Cercozoa, Apusozoa, Myzozoa, Loukozoa) within the basal kingdom Protozoa, mostly in known classes, sometimes even in known orders, families or genera . This strengthens the idea that the ancestral eukaryote was a mitochondrial aerobe . Analogous claims of novel bacterial divisions or kingdoms may reflect the weak resolution and grossly non-clock-like evolution of ribosomal rRNA, not genuine phylum-level biological disparity . Critical interpretation of environmental DNA sequences suggests that our overall picture of microbial biodiversity at phylum or division level is already rather good and comprehensive and that there are no uncharacterized kingdoms of life . However, immense lower-level diversity remains to be mapped, as does the root of the tree of life.

Proc R Soc Lond B Biol Sci, 2004 Jul 22, 271(1547), 1529 - 35
Bacteriocins, spite and virulence; Gardner A et al.; There has been much interest in using social evolution theory to predict the damage to a host from parasite infection, termed parasite virulence . Most of this work has focused on how high kinship between the parasites infecting a host can select for more prudent exploitation of the host, leading to a negative relationship between virulence and parasite kinship . However, it has also been shown that if parasites can cooperate to overcome the host, then high parasite kinship within hosts can select for greater cooperation and higher growth rates, hence leading to a positive relationship between virulence and parasite kinship . We examine the impact of a spiteful behaviour, chemical (bacteriocin) warfare between microbes, on the evolution of virulence, and find a new relationship: virulence is maximized when the frequency of kin among parasites' social partners is low or high, and is minimized at intermediate values . This emphasizes how biological details can fundamentally alter the qualitative nature of theoretical predictions made by models of parasite virulence .

Methods Mol Med, 2004, 101, 93 - 106
Laser-mediated microdissection as a tool for molecular analysis in arthritis; Judex M et al.; Most current approaches to the analysis of gene expression in arthritic tissue samples are based on RNA isolated either from cultured synovial cells or from synovial biopsies . However, this strategy does not distinguish between specific gene expression profiles of cells originating from separate tissue areas . Therefore, we established the combination of laser-mediated microdissection and RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display to analyze profiles of gene expression in histologically defined areas of arthritic tissue . Cryosections derived from synovial tissue were used to obtain cell samples from different tissue areas using a microbeam laser microscope . RNA was isolated and analyzed using nested RAP-PCR to generate a fingerprint of the expressed gene sequences . Differentially expressed bands were isolated, cloned, and sequenced . Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry.

Curr Microbiol, 2004 Jul, 49(1), 10 - 2
Wolbachia replication and host cell division in Aedes albopictus; Ruang-areerate T et al.; Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species . The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing . Like other vertically transmitted intracellular symbionts, Wolbachia's replication rate must not outpace that of its host cells if it is to remain benign . The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis . During diapause in mosquito eggs, no host cell division is believed to occur . Further development is triggered only by subsequent exposure of the egg to water . This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate . We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.

Curr Opin Biotechnol, 2004 Aug, 15(4), 323 - 9
Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts; Becker S et al.; Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme . Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second . The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.

Curr Opin Biotechnol, 2004 Aug, 15(4), 285 - 90
Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms; Streit WR et al.; Modern biotechnology has a steadily increasing demand for vitamins, antibiotics and, in particular, novel biocatalysts for use in the production of flavors, agrochemicals, pharmaceuticals and high-value fine chemicals . Novel experimental approaches are being developed in attempts to identify such molecules . However, it is known that up to 99.8% of the microbes present in many environments are not readily culturable; hence, they cannot be exploited for biotechnology . The 'metagenome technology' offers a solution to this problem by developing culture-independent methods to isolate, clone and express environmental DNA . So far, metagenome-based approaches have led to the isolation of many novel biocatalysts and a variety of other molecules with a high potential for downstream applications.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 227 - 34
From extreme environments to biologically active exopolysaccharides; Guezennec J; In the course of the discovery of novel polysaccharides of biotechnological interest, it is now widely accepted that extremophilic microorganisms will provide a valuable resource not only for exploitation in novel biotechnological processes but also as models for investigating how biomolecules are stabilized when subjected to extreme conditions . Microbes isolated from extreme environments offer a great diversity in chemical and physical properties of their EPS as compared to anywhere else in the biosphere . Bacteria from remote areas still remain virtually unexplored and there is not doubt that extreme environments are a rich source of microorganisms of biotechnological importance . A number of interesting and unique bacterial polysaccharides have been isolated from these ecosystems and are expected to find applications in the very near future in different industrial . Further screenings are underway as well as research into understanding the structure-function relationships of these unusual polymers.

Crit Care Nurs Q, 2004 Jul-Sep, 27(3), 241 - 57
Nosocomial pneumonia; Myrianthefs PM et al.; Nosocomial pneumonia (NP) is defined as pneumonia that develops within 48 hours or more of hospital admission and which was not developing at the time of admission . Nosocomial pneumonia, also known as hospital-acquired pneumonia (HAP), is the second most common hospital infection, while ventilator-associated pneumonia represents the most common intensive care unit (ICU) infection . Nosocomial pneumonia significantly contributes to morbidity, mortality, and escalating healthcare costs because of increases in antibiotic prescription and administration, length of ICU stay, and length of hospital stay . Aspiration and colonization of the upper respiratory tract seem to be the major pathogenetic mechanisms for the development of NP, either in intubated or spontaneously breathing patients . The microbiology of NP depends on the timing of onset . In early-onset NP, the responsible pathogens are generally endogenous community-acquired pathogens . In late-onset NP, the responsible microbes include potentially multi-drug-resistant nosocomial organisms residing in oropharyngeal or gastric contents . Important risk factors for development of NP include coma, intubation, prolonged mechanical ventilation, repeated intubations, supine positioning, and long-term antibiotic use . The most significant preventive measures include routine hand washing and avoidance of (1) the supine position, (2) inappropriate antibiotics, and (3) overuse of H2-antagonists for stress ulcer prophylaxis . Accurate diagnosis of NP is difficult and controversial, warranting consideration for the application of invasive quantitative culture techniques over tracheal aspirates . Empiric antibiotic treatment should be prompt, starting on clinical suspicion, and based on local ICU pathogen epidemiology and antibiotic resistance patterns and on a deescalating antibiotic strategy . Innovative antibiotic strategies, such as antibiotic rotation, to help prevent the emergence of multi-drug-resistant pathogens and improve survival should be considered.

Biol Reprod, 2004 Dec, 71(6), 1828 - 37 Epub 2004 Dec.
Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa; Paasch U et al.; We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa . Eleven pools of cryopreserved and eight pools of fresh semen samples were examined . Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB) . Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay . Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation . In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels . In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim . These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.

Phys Med Biol, 2004 Jul 7, 49(13), N203 - 4
Uniaxial and biaxial irradiation protocols for microbeam radiation therapy; Slatkin DN; Synchrotron-generated x-ray beams for microbeam radiation therapy (MRT) are fixed in space, so three-dimensional treatment planning would require that a patient be secured to, and moved in a gantry between exposures . Two protocols for such movements are proposed: one for uniaxial opposing-fields cross-planar MRT, the other for biaxial orthogonal-fields co-planar MRT.

J Nutr, 2004 Aug, 134(8 Suppl), 2017S - 2021S
The stratum corneum barrier: the final frontier; Marks R; The stratum corneum (SC) is the differentiated end product of the mammalian epidermis . It is vital to constancy of the milieu interieur (the environment within) because it prevents water loss and the penetration by potentially toxic xenobiotics, damaging radiation, and pathogenic microbes . The intercorneocyte space contains complex nonpolar lipids that constitute the water barrier . The formation of the SC in the process of keratinization is complex providing multiple opportunities for disorders to arise . The final act of keratinization is desquamation and for this to occur the controlled release of single corneocytes is required in which proteases play an important role . Tests and techniques are described that measure the structure and function of the SC.

Med Mycol, 2004 Jun, 42(3), 189 - 216
A vaccine against coccidioidomycosis is justified and attainable; Cole GT et al.; Coccidioides is a fungal pathogen of humans which can cause a life-threatening respiratory disease in immunocompetent individuals . Recurrent epidemics of coccidioidal infections in Southwestern United States has raised the specter of awareness of this soil-borne microbe, particularly among residents of Arizona and Southern California, and has galvanized research efforts to develop a human vaccine against coccidioidomycosis . In this review, we discuss the rationale for such a vaccine, examine the features of host innate and acquired immune response to Coccidioides infection, describe strategies used to identify and evaluate vaccine candidates, and provide an update on progress toward development of a vaccine against this endemic pathogen.

J Appl Microbiol, 2004, 97(3), 640 - 6
Screening for novel laccase-producing microbes; Kiiskinen LL et al.; AIMS: To discover novel laccases potential for industrial applications . METHODS AND RESULTS: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions . The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid . The screening work resulted in isolation of 26 positive fungal strains . Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening . Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI . The laccases showed good thermal stability at 60 degrees C . CONCLUSIONS: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers . The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478 . SIGNIFICANCE AND IMPACT OF THE STUDY: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification . It is essential to find novel, efficient enzymes to further develop these applications . This study showed that relatively simple plate test screening method can be used for discovery of novel laccases .

Biotechnol Appl Biochem, 2004 Dec, 40(Pt 3), 243 - 53
Evaluation of the diffusion coefficient for controlled release of oxytetracycline from alginate/chitosan/poly(ethylene glycol) microbeads in simulated gastrointestinal environments; Cruz MC et al.; Diffusion studies of OTC (oxytetracycline) entrapped in microbeads of calcium alginate, calcium alginate coacervated with chitosan (of high, medium and low viscosity) and calcium alginate coacervated with chitosan of low viscosity, covered with PEG {poly(ethylene glycol) of molecular mass 2, 4.6 and 10 kDa, were carried out at 37+/-0.5 degrees C, in pH 7.4 and pH 1.2 buffer solutions - conditions similar to those found in the gastrointestinal system . The diffusion coefficient, or diffusivity (D), of OTC was calculated by equations provided by Crank {(1975) Mathematics in Diffusion, p . 85, Clarendon Press, Oxford} for diffusion, which follows Fick's {(1855) Ann . Physik (Leipzig) 170, 59} second law, considering the diffusion from the inner parts to the surface of the microbeads . The least-squares and the Newton-Raphson {Carnahan, Luther and Wilkes (1969) Applied Numerical Methods, p . 319, John Wiley & Sons, New York} methods were used to obtain the diffusion coefficients . The microbead swelling at pH 7.4 and OTC diffusion is classically Fickian, suggesting that the OTC transport, in this case, is controlled by the exchange rates of free water and relaxation of calcium alginate chains . In case of acid media, it was observed that the phenomenon did not follow Fick's law, owing, probably, to the high solubility of the OTC in this environment . It was possible to modulate the release rate of OTC in several types of microbeads . The presence of cracks formed during the process of drying the microbeads was observed by scanning electron microscopy.

Indian J Pediatr, 2004 Jul, 71(7), 617 - 25
Standard precautions and post exposure prophylaxis for preventing infections; Sridhar MR et al.; In health care set up, risk of acquiring infection by both patients and health care worker (HCW) from each other is fairly high . Despite progress, hospital acquired infections (HAI) are a problem in both developed and developing countries and are an important cause of death . Many different microbes cause HAI in both patients and HCW; these include various commensals, pathogenic bacteria, viruses, parasites, and fungi . Among these HIV, hepatitis B, and hepatitis C are of major significance to HCW . 'Standard precautions' have now replaced the term 'universal precautions', and are designed to reduce the risk of transmission of microorganisms in health care set-up from both recognized and unrecognized sources . Ultimate aim is to reduce the risk of disease transmission in the healthcare setting, both to the patient and the provider, and thus reduce morbidity . This applies to all patients, regardless of their diagnosis and expands the coverage of universal precautions by recognizing that any body fluid may contain contagious and harmful microorganisms . This article reviews the standard precautions and discusses current guidelines on post exposure prophylaxis (PEP).

Curr Med Chem, 2004 Aug, 11(16), 2161 - 83
The design and development of deferiprone (L1) and other iron chelators for clinical use: targeting methods and application prospects; Kontoghiorghes GJ et al.; Iron is essential for all human cells as well as neoplastic cells and invading microbes . Natural and synthetic iron chelators could affect biological processes involving iron and other metal ions in health and disease states . Iron overload is the most common metal toxicity condition worldwide . There are currently two iron chelating drugs, which are mostly used for the treatment of thalassaemia and other conditions of transfusional iron overload . Deferoxamine was until recently the only approved iron chelating drug, which is effective but very expensive and administered parenterally resulting in low compliance . Deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one) is the world's first and only orally active iron chelating drug, which is effective and inexpensive to synthesise thus increasing the prospects of making it available to most thalassaemia patients in third world countries who are not currently receiving any form of chelation therapy . Deferiprone has equivalent iron removal efficacy and comparable toxicity to deferoxamine . There are at least four other known iron chelators, which are currently being developed . Even if successful, these are not expected to become available for clinical use in the next five years and to be as inexpensive as deferiprone . The variation in the chemical, biological, pharmacological, toxicological and other properties of the chelating drugs and experimental chelators provide evidence of the difference in the mode of action of chelators and the need to identify and select molecular structures and substituents based on structure/activity correlations for specific pharmacological activity . Such information may increase the prospects of designing new chelating drugs, which could be targeted and act on different tissues, organs, proteins and iron pools that play important role not only in the treatment of iron overload but also in other diseases of iron and other metal imbalace and toxicity including free radical damage . Chelating drugs could also be designed, which could modify the enzymatic activity of iron and other metal containing enzymes, some of which play a key role in many diseases such as cancer, inflammation and atherosclerosis . Other applications of iron chelating drugs could involve the detoxification of toxic metals with similar metabolic pathways to iron such as Al, Cu, Ga, In, U and Pu.

Trends Microbiol, 2004 Aug, 12(8), 386 - 93
Unravelling rhizosphere-microbial interactions: opportunities and limitations; Singh BK et al.; The rhizosphere is a biologically active zone of the soil around plant roots that contains soil-borne microbes including bacteria and fungi . Plant-microbe interactions in the rhizosphere can be beneficial to the plant, the microbes or to neither of them . One of the major difficulties that plant biologists and microbiologists face when studying these interactions is that many groups of microbes that inhabit this zone are not cultivable in the laboratory . Recent developments in molecular biology methods are shedding some light on rhizospheric microbial diversity . This review discusses recent findings and future challenges in the study of plant-microbe interactions in the rhizosphere.

Phytochemistry, 2004 Jun, 65(12), 1721 - 32
Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction; Valot B et al.; Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions . Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species . To complement the proteomic data, which have mainly focused on the total root proteins from M . truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins . Following the setting up of the purification process, microsomal proteins were separated on 2-DE . Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting . A high percent (83%) of successful protein identification was obtained when using M . truncatula clustered EST database for queries . During the purification process, the enrichment in membrane-associated proteins was monitored on 2-D gels . The membrane location of microsomal proteins was further confirmed using PMF identification . This study reports a fractionation process for characterizing microsomal root proteins of M . truncatula, which could be an interesting tool for investigating the molecular mechanisms involved in root symbioses.

Parasitol Today, 1996 May, 12(5), 187 - 94
Amphids in strongyloides stercoralis and other parasitic nematodes; Ashton FT et al.; In this review, Francis Ashton and Gerhard Schad examine the ultrastructure of the amphids of several animal parasitic nematodes . These structures are the main chemosensory organs of these worms and probably play an important role in host-finding behavior and the control of development . Reconstructions made from serial micrographs of the neurons in the amphids of the threadworm Strongyloides stercoralis are shown . These stereo images permit three-dimensional visualization of these complex sense organs . The association between each amphidial neuron and its cell body has not been made previously for a parasitic nematode; however, this has been done for the free-living nematode Caenorhabditis elegans, which served as a model for these studies . Recognition of the cell bodies will provide a point of departure for laser microbeam ablation studies to determine individual neuronal function.

J Chem Ecol, 2004 May, 30(5), 1035 - 44
Genetic analysis of benzoquinone production in Tribolium confusum; Yezerski A et al.; Many species of tenebrionid beetles produce and secrete benzoquinones from specialized prothoracic and postabdominal glands . Tribolium confusum produces two compounds methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ) . These compounds are hypothesized to function as external defense compounds, killing microbes and deterring predators, and their ability to evolve by natural selection depends on both selection and the genetic vs . environmental contribution to phenotypic variation . We crossed a strain of T . confusum that produces high quantities of benzoquinones, b-Pakistan, with a low-producing strain, b-+, and measured both the internal and external quantities of MBQ and EBQ for the two extreme strains and their F1 progeny . Internal amounts show a clear pattern of inheritance, with at least 50% of the phenotypic variation attributed to genotype . Additive and dominance coefficients for internal amounts indicate that the trait is additive with no significant dominance . In contrast, external quantities show little pattern of inheritance . The role of genetics and environment in determining quantities of secretory defensive compounds is important to elucidating the ecology and evolutionary potential of chemical defenses.

Oncogene, 2004 Sep 2, 23(40), 6830 - 44
Comparison of gene-expression profiles between diffuse- and intestinal-type gastric cancers using a genome-wide cDNA microarray; Jinawath N et al.; Gastric cancer is the fourth leading cause of cancer-related death in the world . Two histologically distinct types of gastric carcinoma, 'intestinal' and 'diffuse', have different epidemiological and pathophysiological features that suggest different mechanisms of carcinogenesis . A number of studies have investigated intestinal-type gastric cancers at the molecular level, but little is known about mechanisms involved in the diffuse type, which has a more invasive phenotype and poorer prognosis . To clarify the mechanisms that underlie its development and/or progression, we compared the expression profiles of 20 laser-microbeam-microdissected diffuse-type gastric-cancer tissues with corresponding noncancerous mucosae by means of a cDNA microarray containing 23,040 genes . We identified 153 genes that were commonly upregulated and more than 1500 that were commonly downregulated in the tumors . We also identified a number of genes related to tumor progression . Furthermore, comparison of the expression profiles of diffuse-type with those of intestinal-type gastric cancers identified 46 genes that may represent distinct molecular signatures of each histological type . The putative signature of diffuse-type cancer exhibited altered expression of genes related to cell-matrix interaction and extracellular-matrix (ECM) components, whereas that of intestinal-type cancer represented enhancement of cell growth . These data provide insight into different mechanisms underlying gastric carcinogenesis and may also serve as a starting point for identifying novel diagnostic markers and/or therapeutic targets for diffuse-type gastric cancers.

Parazitologiia, 2004 May-Jun, 38(3), 261 - 5
{Experimental study of the infecting ability of the flea Coptopsylla lamellifer rostrata in the Kyzylkum natural focus of plague}; Bliummer AG; Experimental infecting of the fleas Coptopsylla lamellifer rostrata by plague was carried out . The fleas were infected by feeding through biomembrane (skin of a white mouse) with a mixture of defibrinated blood of guinea-pig and plague microbes . Under concentration 2-3 milliard of the microbes in 1 millilitre, from 60 to 100% of the fleas were infected . Forming of the block of proventriculus was observed in 9-13 day . Mean percent of blocking for all experiments was less than 1% . Maximal rate of blocking (2.8%) was observed in the experiment under everyday 3-hour feeding . After planting of the fleas with blocks on great gerbil (2 fleas on one gerbil) 20% of the gerbils were infected . The conclusion had been made that C . lamellifer rostrata has only insignificant effectiveness as a vector of plague agent in Kyzylkum . Probably it is caused by some specific features of this subspecies.

J Environ Sci (China), 2004, 16(3), 520 - 4
Two stages kinetics of municipal solid waste inoculation composting processes; Xi BD et al.; In order to understand the key mechanisms of the composting processes, the municipal solid waste (MSW) composting processes were divided into two stages, and the characteristics of typical experimental scenarios from the viewpoint of microbial kinetics was analyzed . Through experimentation with advanced composting reactor under controlled composting conditions, several equations were worked out to simulate the degradation rate of the substrate . The equations showed that the degradation rate was controlled by concentration of microbes in the first stage . The degradation rates of substrates of inoculation Run A, B, C and Control composting systems were 13.61 g/(kg x h), 13.08 g/(kg x h), 15.671 g/(kg x h), and 10.5 g/(kg x h), respectively . The value of Run C is around 1.5 times higher than that of Control system . The decomposition rate of the second stage is controlled by concentration of substrate . Although the organic matter degradation rates were similar to all Runs, inoculation could reduce the values of the half velocity coefficient Km and could be more efficient to make the composting stable . Particularly, for Run C, the degradation rate is high in the first stage, and Km is low in the second stage . The results indicated that the inoculation was efficient for the composting processes.

Immunology, 2004 Aug, 112(4), 521 - 30
The potential for Toll-like receptors to collaborate with other innate immune receptors; Mukhopadhyay S et al.; Cells of the innate immune system express a large repertoire of germ-line encoded cell-surface glycoprotein receptors including Toll-like receptors (TLRs) . TLRs recognize conserved motifs on microbes and induce inflammatory signals . Evidence suggests that individual members of the TLR family or other non-TLR surface antigens either physically or functionally interact with each other and cumulative effects of these interactions instruct the nature and outcome of the immune response to a particular pathogen.

Biotechnol Appl Biochem, 2004 Aug, 40(Pt 1), 25 - 34
Human adult craniofacial muscle-derived cells: neural-cell adhesion-molecule (NCAM; CD56)-expressing cells appear to contain multipotential stem cells; Sinanan AC et al.; Skeletal muscle has been well characterized as a reservoir of myogenic precursors or satellite cells with the potential to participate in cellular repopulation therapies for muscle dysfunction . Recent evidence, however, suggests that the postnatal muscle compartment can be considered an alternative to bone marrow as a source of multipotent cells or muscle-derived stem cells (MDSCs) . MDSCs, when primed with appropriate environmental cues, can differentiate into a variety of non-muscle cells . The present study describes the application of a new technique for the isolation of adult human myoblasts and putative MDSCs, based on microbead-immunomagnetic selection of CD56+ cells, derived from craniofacial skeletal muscle, and details changes in morphological/molecular phenotype of the purified cells when maintained in either a myogenic or a non-myogenic milieu . Multiple immunofluorescence microscopy and two-colour flow-cytometric analysis of proliferating CD56+ cultures revealed positive staining for myogenic markers (CD56, desmin and M-cadherin) as well as putative stem-cell markers {the antigens CD34, CD90 and CD106, and Flk-1 (fetal liver kinase-1)/VEGFR-2 (vascular-endothelial-growth-factor receptor)} . Confluent cultures subjected to cycles of adipogenic or osteogenic induction contained either adipocytes or osteoblasts and myotubes . In conclusion, the CD56+ subpopulation within adult human skeletal muscle is heterogeneous and is composed of both lineage-committed myogenic cells and multipotent cells (the candidate MDSCs), which are able to form non-muscle tissue such as fat and bone.

J Synchrotron Radiat, 1998 May 1, 5(Pt 3), 1111 - 3 Epub 1998 May 01.
Submicrometre-area high-energy-resolution photoelectron spectroscopy system; Kiyokura T et al.; A submicrometre-area photoelectron spectroscopy system that uses a multi-layer-coated Schwarzschild objective as the soft X-ray microbeam optics has been developed . The system is located at an undulator beamline (BL-16U) at the Photon Factory in the High Energy Accelerator Research Organization . By knife-edge measurement, the microbeam size was estimated to be 160 nm at the sample position using a 25-75% criterion . Photoelectron spectral measurements revealed that the Fermi edge width was 0.12 eV, which means that the instrumental resolution was 0.05 eV, after removing the natural broadening of the Fermi edge at room temperature . This system offers both high energy resolution and high spatial resolution.

J Synchrotron Radiat, 1998 May 1, 5(Pt 3), 902 - 4 Epub 1998 May 01.
Micro X-ray diffraction analysis of thin films using grazing-exit conditions; Noma T et al.; An X-ray diffraction technique using a hard X-ray microbeam for thin-film analysis has been developed . To optimize the spatial resolution and the surface sensitivity, the X-ray microbeam strikes the sample surface at a large glancing angle while the diffracted X-ray signal is detected with a small (grazing) exit angle . Kirkpatrick-Baez optics developed at the Photon Factory were used, in combination with a multilayer monochromator, for focusing X-rays . The focused beam size was about 10 x 10 micro m . X-ray diffraction patterns of Pd, Pt and their layered structure were measured . Using a small exit angle, the signal-to-background ratio was improved due to a shallow escape depth . Under the grazing-exit condition, the refraction effect of diffracted X-rays was observed, indicating the possibility of surface sensitivity.

Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 11025 - 9 Epub 2004 Jul 19.
Resistant tissues of modern marchantioid liverworts resemble enigmatic Early Paleozoic microfossils; Graham LE et al.; Absence of a substantial pretracheophyte fossil record for bryophytes (otherwise predicted by molecular systematics) poses a major problem in our understanding of earliest land-plant structure . In contrast, there exist enigmatic Cambrian-Devonian microfossils (aggregations of tubes or sheets of cells or possibly a combination of both) controversially interpreted as an extinct group of early land plants known as nematophytes . We used an innovative approach to explore these issues: comparison of tube and cell-sheet microfossils with experimentally degraded modern liverworts as analogues of ancient early land plants . Lower epidermal surface tissues, including rhizoids, of Marchantia polymorpha and Conocephalum conicum were resistant to breakdown after rotting for extended periods or high-temperature acid treatment (acetolysis), suggesting fossilization potential . Cell-sheet and rhizoid remains occurred separately or together depending on the degree of body degradation . Rhizoid break-off at the lower epidermal surface left rimmed pores at the centers of cell rosettes; these were similar in structure, diameter, and distribution to pores characterizing nematophyte cell-sheet microfossils known as Cosmochlaina . The range of Marchantia rhizoid diameters overlapped that of Cosmochlaina pores . Approximately 14% of dry biomass of Marchantia vegetative thalli and 40% of gametangiophores was resistant to acetolysis . Pre- and posttreatment cell-wall autofluorescence suggested the presence of phenolic compounds that likely protect lower epidermal tissues from soil microbe attack and provide dimensional stability to gametangiophores . Our results suggest that at least some microfossils identified as nematophytes may be the remains of early marchantioid liverworts similar in some ways to modern Marchantia and Conocephalum.

Biochem J, 2004 Dec 1, 384(Pt 2), 263 - 70
Effects of pH on Helicobacter pylori binding to human gastric mucins: identification of binding to non-MUC5AC mucins; Linden S et al.; Helicobacter pylori causes gastritis, peptic ulcer disease and gastric cancer . The microbe is found in the gastric mucus layer where a pH gradient ranging from acidic in the lumen to neutral at the cell surface is maintained . The aim of the present study was to investigate the effects of pH on H . pylori binding to gastric mucins from healthy individuals . At pH 3, all strains bound to the most charged MUC5AC glycoform and to a putative mucin of higher charge and larger size than subunits of MUC5AC and MUC6, irrespective of host blood-group . In contrast, at pH 7.4 only Le(b)-binding BabA-positive strains bound to Le(b)-positive MUC5AC and to smaller mucin-like molecules, including MUC1 . H . pylori binding to the latter component(s) seems to occur via the H-type-1 structure . All strains bound to a proteoglycan containing chondroitin sulphate/dermatan sulphate side chains at acidic pH, whereas binding to secreted MUC5AC and putative membrane-bound strains occurred both at neutral and acidic pH . The binding properties at acidic pH are thus common to all H . pylori strains, whereas mucin binding at neutral pH occurs via the bacterial BabA adhesin and the Le(b) antigen/related structures on the glycoprotein . Our work shows that microbe binding to membrane-bound mucins must be considered in H . pylori colonization, and the potential of these glycoproteins to participate in signalling events implies that microbe binding to such structures may initiate signal transduction over the epithelial layer . Competition between microbe binding to membrane-bound and secreted mucins is therefore an important aspect of host-microbe interaction.

J Chem Ecol, 2004 Apr, 30(4), 703 - 17
Semiochemicals from fungal associates of bark beetles may mediate host location behavior of parasitoids; Sullivan BT et al.; In laboratory olfactometer bioassays, females of two hymenopteran parasitoid species, Roptrocerus xylophagorum (Ratzeburg) (Hymenoptera: Pteromalidae) and Spathius pallidus (Ashmead) (Hymenoptera: Braconidae), were attracted to odors from bark or bolts of loblolly pine, Pinus taeda L., colonized by bluestain fungi (genus Ophiostoma) associated with the parasitoids' bark beetle hosts . Mock-inoculated bolts and bark were less attractive or unattractive in these bioassays . Bark infested with host larvae that lacked their fungal and other normal microbial associates was attractive to R . xylophagorum females, but was less so than bark infested with larvae possessing their normal complement of associated microbes . In contrast, in oviposition bioassays, R . xylophagorum females spent approximately equal time searching, made similar numbers of oviposition attempts, parasitized similar percentages of hosts, and laid similar numbers of eggs in bark fragments infested with either associate-free or associate-bearing host larvae . Furthermore, in field bioassays using bluestain-inoculated or mock-inoculated loblolly pine bolts as sources of attractants, the numbers of parasitoids attracted by the two treatments did not differ significantly and the two treatments were less attractive than bolts naturally infested with bark beetle larvae . Whereas our laboratory olfactometer data suggest that bark beetle fungal associates may enhance attraction of some parasitoids, our bioassays with associate-free hosts indicate that associate-produced are not required for short-range host location and parasitization . In addition, our field trials indicated that long-range attraction of parasitoids to the host-fungi-tree complex is not caused simply by an interaction between bluestain fungi and tree tissues.

Commun Dis Public Health, 2004 Jun, 7(2), 152 - 3
The behaviour of microbes; Clewley JP; Viruses and bacteria have complex interactions with their hosts, beyond mere replication in them . They range from those that are detrimental, to others that may be non-pathogenic or even beneficial . Molecular techniques can help to unravel these interactions, sometimes revealing phenomena that benefit host as well as microbial populations.

Cell Tissue Bank, 2001, 2(3), 127 - 33
Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting; Grunewald S et al.; Cryopreservation increases the rate of apoptotic spermatozoa with decreased capability to fertilise oocytes . In order to optimise the fertilisation rates, especially in assisted reproduction the use of apoptotic sperms should be avoided . Early events of apoptosis in cryopreserved spermatozoa are not detectable by conventional methods . However, the surface of apoptotic spermatozoa is characterised by externalisation of phosphatidylserine (PS), which has a high affinity to Annexin V . Therefore, colloid paramagnetic Annexin-V-conjugated microbeads (AN-MB) were tested for their ability to eliminate apoptotic spermatozoa from a total of 40 fresh and in TEST yolk buffer cryopreserved semen samples which were provided by 15 healthy volunteers . By passing through a magnetic field (MiniMACS, Miltenyi Biotec) the sperm suspensions were divided into 2 sperm fractions depending on bound magnetic Annexin V-microbeads (AN-MB) to spermatozoa . As additional markers of apoptosis CD95 (Fas, APO-1) on the sperm surface and activated caspases in the cytosol were detected in both fractions . Supplementary investigations comprised eosin-supravital staining and computer assisted sperm motion analysis . The separation was supervised by flow cytometric analysis of spermatozoa labelled with FITC-conjugated anti Annexin V-antibodies . Analyses of the magnetic inactive sperm fraction (AN-MB-negative) showed CD95 on 0.6 +/- 0.3% (X +/- SEM) of spermatozoa and only 3.2 +/- 0.5% were stainable with eosin, whereas, 40.6 +/- 6.7% of the remaining cells in the column appeared to be CD95 positive and 99.8 +/- 0.1% stainable with eosin after cryopreservation . Indeed the overall amount of CD95 positive spermatozoa did not significantly increase after cryopreservation (2.5 +/- 0.5% vs . 4.3 +/- 1.2%; p > 0.05) . Activated caspases were found in 21.8 +/- 2.6% of the spermatozoa in fresh and in 47.7 +/- 5.8% of cryopreserved semen samples (p < 0.01) . The separation procedure of the cryopreserved spermatozoa reduced significantly the quantity of those containing activated caspases to 9.3 +/- 2.2% within the AN-MB-negative fraction . In contrast 89.1 +/- 2.3% of AN-MB-positive sperms showed activation of these proteolytic enzymes . Flow cytometric analyses using FITC-conjugated anti Annexin V-antibodies for monitoring of AN-MB-binding to spermatozoa showed 5.2 +/- 1.0% labelled spermatozoa in the AN-MB negative fraction and 72.6 +/- 2.7% labelled spermatozoa in the AN-MB positive one . There was no significant influence of the separation column and the magnetic field on the sperm functions . The passage through the column led to a sperm loss of 0.8 +/- 1.2% . Conclusion: The binding of paramagnetic Annexin V-conjugated microbeads is an excellent method to eliminate spermatozoa at early apoptotic stages from cryopreserved semen samples . A deleterious influence of the separation column and the magnetic field on the spermatozoa was not observed.

Public Health Rev, 2003, 31(2), 81 - 91
The impact of "ancient pathogen" studies on the practice of public health; Greenblatt C et al.; A new field of "ancient pathogens" is making an impact on our concepts of the evolution of infectious diseases, and it will eventually alter the practice of public health in their control . It has begun to answer important questions regarding past epidemics of influenza and tuberculosis by recovering the genetic sequences of the ancient causative agents . Vaccination strategics will have to study these microbial variants in order to develop tomorrow's vaccines . It may also be possible to examine the role of past and present reservoirs in the dynamics of emerging diseases . In unraveling the evolution of pathogens, insights into the mechanisms of drug and antibiotic resistance are possible . As "genome projects" of more and more pathogens are being completed . Targets for chemotherapy are being revealed which are totally different from the metabolic processes of the mammalian host . Signal molecules are being identified which alter the virulence of the microbe . Focussing on these mechanisms without attempting to kill the pathogen may in some cases drive it into a benign state . These and other aspects of the evolution of pathogens are discussed which may lead to innovative approaches to the control of infectious diseases.

Astrobiology, 2004 Summer, 4(2), 196 - 210
A transmission electron microscopy study of silica and kerogen biosignatures in approximately 1.9 Ga gunflint microfossils; Moreau JW et al.; Microfossils preserved in chert from the;1.9 Ga Gunflint Formation (Schreiber Beach, Ontario, Canada) were studied with transmission electron microscopy (TEM) and analytical TEM (ATEM) . Our goals were to uncover the style of silicification relative to the distribution of organic matter, and to evaluate the distribution and evolution of organic matter, at submicroscopic spatial scales . Petrographically the microfossils typically display filamentous or coccoidal morphologies, and consist of quartz crystals surrounded by kerogen along grain boundaries . ATEM analysis revealed that quartz associated with kerogen consists of 200-500nm-sized, round crystallites, whereas the chert matrix is comprised of randomly oriented, polygonal microquartz (5-10 microm) . Silica spheroids found within some fossils consist of quartz subgrains in an amorphous to poorly crystalline matrix, suggesting that precipitation of opaline silica on organic matter occurred with subsequent but incomplete transformation to quartz . Some coccoidal microfossils surround large euhedral quartz crystals (up to 5 microm in diameter) that appeared to have influenced the distribution of kerogen during crystal growth . These euhedral quartz crystals commonly contain elongated (50-100 nm) iron-rich crystallites . Energy-loss, near-edge structure analysis of kerogen associated with a coccoidal microfossil showed that it is composed of amorphous carbon with no evidence of graphitization . TEM results revealed significant differences in the style of silicification between microbe-shaped microfossils and their surrounding chert matrix, as well as the presence of amorphous kerogen.

Int Microbiol, 2004 Jun, 7(2), 83 - 94
Recombinant viruses as tools to induce protective cellular immunity against infectious diseases; Rocha CD et al.; Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells . Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms . Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen . However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes . In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools . Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes . This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development . Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.

Autoimmun Rev, 2004 Jun, 3(4), 295 - 300
Sarcoidosis succumbs to antibiotics--implications for autoimmune disease; Marshall TG et al.; From time to time there have been reports of autoimmune disease succumbing to tetracycline antibiotics, but many have assumed this was due to coincidence, or to some ill-defined 'anti-inflammatory property' of the tetracyclines . But now the inflammation of sarcoidosis has succumbed to antibiotics in two independent studies . This review examines the cell wall deficient (antibiotic resistant) bacteria which have been found in tissue from patients with sarcoidosis . It examines how such bacteria can infect the phagocytes of the immune system, and how they may therefore be responsible for not only sarcoid inflammation, but also for other autoimmune disease . Proof positive of a bacterial pathogenesis for Sarcoidosis includes not only the demonstrated ability of these studies to put the disease into remission, but also the severity of Jarisch-Herxheimer shock resulting from endotoxin release as the microbes are killed . Studies delineating the hormone responsible for phagocyte differentiation in the Th1 immune response, 1,25-dihydroxyvitamin D, are discussed, and its utility as a marker of Th1 immune inflammation is reviewed . Finally, data showing that the behavior of this hormone is also aberrant in rheumatoid arthritis, systemic lupus erythematosus, and Parkinson's, raise the possibility that these diseases may also have a CWD bacterial pathogenesis.

Curr Opin Immunol, 2004 Aug, 16(4), 483 - 7
Toll-like receptors and microbes take aim at each other; Underhill DM; Toll-like receptors (TLRs) are key components of effective innate immunity . As such, pathogenic microorganisms must evade recognition by TLRs, manipulate the consequences of TLR activation, or contend with the inflammatory consequences of TLR activation . Recent studies have improved our understanding of the various mechanisms by which pathogenic organisms manipulate the TLR recognition system.

Indian J Exp Biol, 2003 Oct, 41(10), 1124 - 32
Cysteine proteases in nodulation and nitrogen fixation; Sheokand S et al.; The cysteine proteinases or cysteine endopeptidases (EC 3.4.22) are known to occur widely in plant cells . They are involved in almost all aspects of plant growth and development including germination, circadian rhythms, senescence and programmed cell death . They are also involved in mediating plant cell responses to environmental stress (such as water stress, salinity, low temperature, wounding, ethylene, and oxidative conditions) and plant-microbe interactions (including nodulation) . In the development and function of legume root nodules, cysteine proteases could be involved in several important processes:-(i) a defence response to root invasion by microorganisms; (ii) protein turnover required during the formation of new tissue; (iii) cellular homeostasis and metabolism; (iv) adaptation of host cells to physiological stresses; (v) control of nodule senescence . Because of their central importance to plant physiology, cysteine proteases could serve as important targets for the study of nodule development and functioning at the molecular level . Because of their widespread occurrence in nodulating plants they could also serve as candidate genes for targeted plant breeding programmes.

Nature, 2004 Jul 8, 430(6996), 257 - 63
Inferences, questions and possibilities in Toll-like receptor signalling; Beutler B; The Toll-like receptors (TLRs) are the key proteins that allow mammals--whether immunologically naive or experienced--to detect microbes . They lie at the core of our inherited resistance to disease, initiating most of the phenomena that occur in the course of infection . Quasi-infectious stimuli that have been used for decades to study inflammatory mechanisms can activate the TLR family of proteins . And it now seems that many inflammatory processes, both sterile and infectious, may depend on TLR signalling . We are in a good position to apply our understanding of TLR signalling to a range of challenges in immunology and medicine.

Nature, 2004 Jul 8, 430(6996), 250 - 6
Frontal and stealth attack strategies in microbial pathogenesis; Merrell DS et al.; Interactions between microbes and human hosts can range from a benign, even symbiotic collaboration to a competition that may turn fatal--resulting in death of the host, the microbe or both . Despite advances that have been made over the past decades in understanding microbial pathogens, more people worldwide still die every year from infectious disease than from any other cause . This highlights the relevance of continuing to probe the mechanisms used by microorganisms to cause disease, and emphasizes the need for new model systems to advance our understanding of host-pathogen interactions.

Appl Environ Microbiol, 2004 Jul, 70(7), 4216 - 21
Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays; Warsen AE et al.; We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides . Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer . PCR products (ca . 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray . Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546 . This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens . With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA . Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

Circulation, 2004 Jul 20, 110(3), 349 - 55 Epub 2004 Jul 06.
Improvement of postnatal neovascularization by human adipose tissue-derived stem cells; Miranville A et al.; BACKGROUND: Several studies have suggested that stem cells are present in the stroma-vascular fraction (SVF) of adipose tissue (AT) . METHODS AND RESULTS: To characterize the cell populations that compose the SVF of human AT originating from subcutaneous and visceral depots, fluorescence-activated cell sorter analysis was performed by use of fluorescent antibodies directed against the endothelial and stem cell markers CD31, CD34, CD133, and ABCG2 . The freshly harvested SVF contained large numbers of CD34+ cells as well as cells expressing CD133 and ABCG2 . Further analysis of the CD34+ cells revealed 2 CD34+ cell populations with differential expression of the endothelial cell marker CD31 . Selection of the CD34+/CD31- cells by use of magnetic microbeads, followed by cell culture, demonstrated that this cell population could differentiate under appropriate conditions into endothelial cells . Moreover, in mouse ischemic hindlimb, intravenous injection of CD34(+)/CD31(-) cells was associated with an increase in the blood flow and the capillary density and an incorporation of the cells in the leg vasculature . CONCLUSIONS: Our data indicate the presence of a cell population within the SVF of human AT characterized as CD34+/CD31- exhibiting characteristics of endothelial progenitor cells . Therefore, human AT might represent a source of stem/progenitor cells useful for cell therapy to improve vasculogenesis in adults.

Ann Agric Environ Med, 2004, 11(1), 85 - 9
Time of farmers' exposure to biological factors in agricultural working environment; Molocznik A; Working time in conditions of exposure to hazardous factors is an important element indispensable for the evaluation of human exposure in the working environment . Agricultural work is accompanied by co-occurrence of many risk factors threatening farmers' health, e.g . dust, elements of the thermal environment, noise, vibration, chemical and biological agents . Biological factors cause diseases with contagious, allergic or immuno-toxic backgrounds which constitute the majority of farmers' occupational diseases registered in Poland . Exposure to hazardous factors in agricultural working environment is due to contact with plants, animals and organic wastes, more precisely--with microbes, plant and animal particles present in aerogenic agricultural dust, as well as pathogens of contagious and invasive diseases present in contaminated soil, water and plants . Data concerning the duration of farmers' exposure to biological and other factors of the working environment were obtained with the use of the Private Farmer Work Chart . Time-schedule observations concerned an annual work cycle . The study covered 30 farms with the following production profiles: plant (Group A), animal (Group B) and mixed production (Group C) . The total working time was: in Group A - from 106-163 % of the legal working time; in Group B - from 75-147 %; in Group C - from 136-167 % . Among 48 work activities contributing to the full working cycle among the farmers examined, 15 activities were accompanied by 5 factors . These were mainly field activities which covered plant harvesting and fertilizing, chemical plant protection, as well as cultivation activities . Agricultural dust and elements of the thermal environment were the environmental factors most frequently accompanying agricultural work, followed by contact with biological factors, noise, vibration, and chemical agents . Biological factors are a specific element associated with 19 work activities, mainly the spreading of manure, animal breeding and plant harvesting . Farmers' working time in conditions of exposure to these factors in the group of plant producers was 51 % of the total working time on average, among animal breeders--80 % and in the case of mixed production--77 %.

Microsc Microanal, 2004 Jun, 10(3), 324 - 35
On the role of characterization in the design of interfaces in nanoscale materials technology; Ringer SP et al.; This work reviews recent research on the design and control of interfaces in engineering nanomaterials . Four case studies are presented that demonstrate the power of a multimodal approach to the characterization of different types of interfaces . We have used a combination of conventional, high resolution, and analytical transmission electron microscopy, microbeam electron diffraction, and three-dimensional atom probe to study polymer-clay nanocomposites, turbine rotor steels used for power generation, multicomponent aluminum alloys, and nanocrystalline magnetic materials.

Immunol Rev, 2004 Jun, 199, 227 - 50
Modulating vaccine responses with dendritic cells and Toll-like receptors; Pulendran B; The immune system is ignorant or even unresponsive to most foreign proteins that are injected in a soluble, deaggregated form, but when injected together with an immune-stimulating agent (i.e . an adjuvant, such as CpG-rich DNA), these foreign proteins can generate robust immunity and long-lived memory to the antigen . In fact, the nature of the adjuvant is what determines the particular type of immune response that follows, which may be biased towards cytotoxic T-cell responses, antibody responses, particular classes of T-helper responses, or antibody isotypes . Clearly, the ability of a vaccine to skew the response toward a particular type is of paramount importance, because different pathogens require distinct types of protective immunities . Therefore, the quest to manipulate the immune system to generate optimally effective immunity against different pathogens can justifiably be considered the 'grand challenge' of modern immunology . Central to this issue is a rare but widely distributed network of cells known as dendritic cells (DCs) . DCs, which have been called 'Nature's adjuvants,' express pathogen recognition receptors, such as the Toll-like receptors (TLRs) and C-type lectins, which enable them to sense and respond to microbes or vaccines . Research in the last decade has demonstrated a fundamental role for DCs in initiating and controlling the quality and strength of the immune response . As such, DCs and TLRs represent attractive immune modulatory targets for vaccinologists . The present review provides a summary of emerging themes in the biology DCs and TLRs, with a particular focus on relevance for vaccine development.

Immunol Rev, 2004 Jun, 199, 156 - 80
DNA fusion gene vaccines against cancer: from the laboratory to the clinic; Stevenson FK et al.; Vaccination against target antigens expressed by cancer cells has now become a realistic goal . DNA vaccines provide a direct link between identification of genetic markers in tumors and vaccine formulation . Simplicity of manufacture facilitates construction of vaccines against disease subsets or even for individual patients . To engage an immune system that exists to fight pathogens, we have developed fusion gene vaccines encoding tumor antigens fused to pathogen-derived sequences . This strategy activates high levels of T-cell help, the key to induction and maintenance of effective immunity . We have dissected the immunogenic tetanus toxin to obtain specific sequences able to activate antibody, CD4+, or CD8+ T cells to attack selected fused tumor antigens . Principles established in preclinical models are now being tested in patients . So far, objective immune responses against idiotypic antigen of neoplastic B cells have been observed in patients with B-cell malignancies and in normal transplant donors . These responses provide a platform for testing physical methods to improve DNA delivery and strategies to boost responses . For cancer, demands are high, because vaccines have to activate powerful immunity against weak antigens, often in a setting of immune damage or tolerance . Vaccination strategies against cancer and against microbes are sharing knowledge and technology for mutual benefit.

Ultramicroscopy, 2004 Aug, 100(3-4), 203 - 10
Quantitative analysis of the number of antigens immobilized on a glass surface by AFM; Kim H et al.; To develop force measurements using an atomic force microscope (AFM) in a quantitative manner, it is necessary to estimate the number density of target molecules on a sample surface, and for this, the sensitivity of detection should be known . In this study, the AFM was used as a mechanical detector and an antigen and its antibody were used as a model to evaluate the sensitivity of detection . Antigens were immobilized on a glass surface and number density was estimated by monitoring optical absorbance due to product formation by the reaction of crosslinkers . The concentration of antigen was controlled by mixing control peptides . A microbead was used as a probe and antibodies were immobilized on the bead . AFM force measurements were then made for a range of number densities in the order of 10-10(6) antigen molecules per square micrometer of surface and were compared to evaluate the sensitivity of detection . Our result establishes the reliability of estimating a number of molecules like receptors on the cell surface, and indicates that the AFM is useful as a mechanical detector with high sensitivity.

Curr Opin Plant Biol, 2004 Aug, 7(4), 372 - 6
Signaling between nematodes and plants; Bird DM; After hatching in the soil, root-knot nematodes must locate and penetrate a root, migrate into the vascular cylinder, and establish a permanent feeding site . Presumably, these events are accompanied by extensive signaling between the nematode parasite and the host . Hence, much emphasis has been placed on identifying proteins that are secreted by the nematode during the migratory phase . Further progress in understanding the signaling events has been made recently by studying the host response . Striking parallels can be drawn between the nematode-plant interaction and plant symbioses with other microorganisms, and evidence is emerging to suggest that nematodes acquired components of their parasitic armory from those microbes.

Mol Microbiol, 2004 Jul, 53(2), 373 - 80
Channels in microbes: so many holes to fill; Kung C et al.; Among players in neurobiology, ion channels are the demigods that underlie all our senses, behaviour and intelligence . In animals, these 'gated pores' detect ligands, voltage, heat or stretch forces and emit electric or ionic signals . Patch clamp and genome sequencing now show that nearly all microbes also have these 'smart' molecules . Microbial channel proteins have yielded crystal structures so dear to neuroscientists . However, their natural roles in microbial physiology remain largely unknown . The intellectual and technical schisms between 'neuro' and 'micro' biology must be bridged before we know how we became so smart, and whether microbes are just as smart.

Ying Yong Sheng Tai Xue Bao, 2004 Mar, 15(3), 463 - 8
{Microbial characteristics of straw-amended degraded soils in central Tibet and its effect on soil fertility}; Cai X et al.; This paper studied the dynamics of microbial communities in degraded soils of central Tibet under straw amendment and its effect on soil fertility . The results showed that straw amendment, and soil covering with spring highland barley (Hordeum vulgare var . nudum) straw in particular, could supply a beneficial soil environment for the relatively balanced growth of soil microbes, e.g., reduced water evaporation, appropriate temperature, and increased content of organic matter and nutrients . All the results demonstrated the possibility of straw amendment in recovering soil fertility and in reconstructing of soil structure in a relatively short period.

Ying Yong Sheng Tai Xue Bao, 2004 Mar, 15(3), 420 - 4
{Effect of grazing on sandy grassland ecosystem in Inner Mongolia}; Zhao H et al.; This experiment was carried out for 5 years in Horqin sandy land, lnner Mongolia, which had 4 treatments: Non-grazing (NG), light grazing (LG), moderate grazing (MG) and over grazing (OG) . The results showed that different grazing intensities resulted in different development trend of the pasture ecosystem, of which, the injury of OG on pasture ecosystem was very great . The plant diversity, vegetation coverage, plant height and primary productivity under continuous overgrazing for 5 year were 87.9%, 82.1%, 94.0% and 57.0%, respectively, lower than those in NG . The biomass on the OG pasture was only 2.1% of NG, and the contents of soil clay, C and N as well as the quantities of soil microbes and small animals in OG were respectively 6.0%, 31.9%, 25.0%, 95.0% and 75.9% lower than those in NG, but the soil hardness was raised by 274.0% . Especially, the secondary productivity of the pasture became negative from the third year, and the productive foundation of the pasture ecosystem was completely destroyed . Non-grazing was beneficial to pasture, and enclosure caused an increase in vegetation coverage, plant height and primary productivity . The vegetation coverage, plant height and soil status in LG and MG were not as good as those in NG, but were stable and didn't show worsening trend . Based on the above results, it's considered that on the sandy pasture in the semi-arid area of Inner Mongolia, the rational grass utilization ratio is 45%-50%, and the suitable loading capacity is 3-4 sheep unit.hm-2.

Folia Microbiol (Praha), 2004, 49(2), 105 - 11
Microbial community structure along an altitude gradient in three different localities; Ma X et al.; The microbial community structure along an altitude gradient was investigated in different localities, in Kalasi lake, Urumqi river and Sangong river, Xingjiang (China) . The mean numbers of DAPI (4',6-diamidino-2-phenylindole)-stained cells were lower in Kalasi lake than that in Urumqi river and Sangong river; these differences were attributed to increasing environmental harshness including lower soil organic carbon and nitrogen content, more acidic pH and lower annual temperature . In each locality, the numbers of bacteria and archaea measured with two fluorescence-labeled 16S rRNA oligonucleotide probes (EUB338 and ARCH915) were higher in a coniferous forest and lower in desert vegetation . A significant and positive relationship was found between microbial and soil organic carbon and total nitrogen along the altitudinal gradient, indicating that plant communities and soil nutrients influence the soil microbial structure . The results show that the microbial population in higher latitudinal site was fewer than lower latitudinal one, soil microorganisms were positively correlated to soil organic carbon and total nitrogen, and plant communities had an obviously impact on soil microbes.

Sci STKE . 2004 Jun 22;2004(239):re9.
The domains of apoptosis: a genomics perspective; Reed JC et al.; Apoptosis plays important roles in many facets of normal physiology in animal species, including programmed cell death associated with fetal development or metamorphosis, tissue homeostasis, immune cell education, and some aspects of aging . Defects in the regulation of apoptosis contribute to multiple diseases associated with either inappropriate cell loss or pathological cell accumulation . Host-pathogen interactions have additionally provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses . To a large extent, the apoptosis machinery can be viewed as a network, with different nodes connected by physical interactions of evolutionarily conserved domains . These domains can serve as signatures for identification of proteins involved in the network . In particular, the caspase recruitment domains (CARDs); death effector domains (DEDs); death domains (DDs); BIR (baculovirus IAP repeat) domains of inhibitor of apoptosis proteins (IAPs); Bcl-2 family proteins; caspase protease domains; and endonuclease-associated CIDE (cell death-inducing DFF45-like effector) domains are found in common in proteins involved in apoptosis . In the genomes of mammals, genes encoding proteins that carry one or more of these signature domains are often present in multiple copies, making up diverse gene families that permit tissue-specific and highly regulated control of cell life and death decisions through combinations of stimulus-specific gene expression and complex protein interaction networks . In this Review, we organize the repertoire of apoptosis proteins of humans into domain families, drawing comparisons with homologs in other vertebrate and invertebrate animal species, and discuss some of the functional implications of these findings.

Transfusion, 2004 Jul, 44(7), 1087 - 97
Clinical isolation and functional characterization of cord blood CD133+ hematopoietic progenitor cells; Bonanno G et al.; BACKGROUND: Human cord blood is a relevant source of CD133+ HPCs . Clinical-scale isolation of human umbilical cord blood (UCB) CD133+ HPCs using immunomagnetic microbeads and the CliniMACS clinical cell isolator is reported . CD133+ HPCs isolated after large-scale processing were functionally characterized . STUDY DESIGN AND METHODS: Closed disposable sets were used to process nine different samples of RBC-reduced UCB nucleated cells . In-vitro hematopoietic assays and human xenografts in NOD/SCID mice were performed to assess the functional properties of isolated CD133+ cells . Different mixtures of human cytokines were tested for the ability to expand nascent CD133+ HPCs . Furthermore, freshly isolated CD133+ cells were conditioned in culture medium specifically tested to support in-vitro myogenesis or osteogenesis . RESULTS: Isolation procedures yielded the recovery of an average of 2.53 x 10(6) CD133+ HPCs with a mean recovery of 96 percent (referred to as RBC-reduced samples) and a final sample purity of 82 percent . Purified CD133+ cells had high cloning efficiency, had relevant long-term activity, and were capable of repopulating irradiated NOD/SCID mice . In 10-day stroma-free cultures, a 2-fold and 8.3-fold expansion of colony-forming cells (CFCs) and extended long-term culture-initiating cells, respectively, was obtained . Freshly isolated CD133+ cells differentiated into large nucleated cells expressing either myosin D or osteopontin (as revealed by RT-PCR and immuno-cytochemistry), with a protein/mRNA expression comparable to or even higher than that observed in UCB CD133- nucleated cells in identical culture conditions . CONCLUSION: Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primitive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.

Biophys Chem, 2004 Jul 1, 110(1-2), 59 - 72
The persistence exponent of DNA; Poland D; Using the complete genome of Thermoplasma volcanium, as an example, we have examined the distribution functions for the amount of C or G in consecutive, non-overlapping blocks of m bases in this system . We find that these distributions are very much broader (by many factors) than those expected for a random distribution of bases . If we plot the widths of the C-G distributions relative to the widths expected for random distributions, as a function of the block size used, we obtain a power law with a characteristic exponent . The broadening of the C-G distributions follows from the empirical finding that blocks containing a given C-G content tend to be followed by blocks of similar C-G content thus indicating a statistical persistence of composition . The exponent associated with the power law thus measures the strength of persistence in a given DNA . This behavior can be understood using Mandelbrot's model of a fractional Brownian walk . In this model there is a hierarchy of persistence (correlation between blocks) between all parts of the system . The model gives us a way to scale the C-G distributions such that all these functions are collapsed onto a master curve . For a fractional Brownian walk, the fractal dimension of the C-G distribution is simply related to the persistence exponent for the power law . The persistence exponent for T . volcanium is found to be gamma = 0.29 while for a 10 million base segment of the human genome we obtain gamma = 0.39, similar to but not identical with the value found for the microbe.

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