Microorganisms

Oncogene, 2003 Dec 4, 22(55), 8912 - 23
Segregation of NF-kappaB activation through NEMO/IKKgamma by Tax and TNFalpha: implications for stimulus-specific interruption of oncogenic signaling; Iha H et al.; Nuclear factor-kappaB essential modulator (NEMO), also called IKKgamma, has been proposed as a 'universal' adaptor of the I-kappaB kinase (IKK) complex for stimuli such as proinflammatory cytokines, microbes, and the HTLV-I Tax oncoprotein . Currently, it remains unclear whether the many signals that activate NF-kappaB through NEMO converge identically or differently . We have adopted two approaches to answer this question . First, we generated and targeted intracellularly three NEMO-specific monoclonal antibodies (mAbs) . These mAbs produced two distinct intracellular NF-kappaB inhibition profiles segregating TNFalpha from Tax activation . Second, using NEMO knockout mouse fibroblasts and 10 NEMO mutants, we found that different regions function in trans either to complement or to inhibit dominantly TNFalpha, IL-1beta, or Tax activation of NF-kappaB . For instance, NEMO (1-245 amino acids) supported Tax-mediated NF-kappaB activation, but did not serve TNFalpha- or IL-1beta signaling . Altogether, our findings indicate that while NEMO 'universally' adapts numerous NF-kappaB activators, it may do so through separable domains . We provide the first evidence that selective targeting of NEMO can abrogate oncogenic Tax signaling without affecting signals used for normal cellular metabolism.

Hum Pathol, 2003 Nov, 34(11), 1185 - 92
Cutaneous manifestations of Crohn's disease, its spectrum, and its pathogenesis: intracellular consensus bacterial 16S rRNA is associated with the gastrointestinal but not the cutaneous manifestations of Crohn's disease; Crowson AN et al.; The classic pathology of skin disease discontinuous from the inflamed gastrointestinal (GI) tract in patients with Crohn's disease (CD) includes pyoderma gangrenosum (PG), erythema nodosum (EN), and so-called metastatic Crohn's disease . The purpose of this study was two-fold: First, we explored the full spectrum of cutaneous lesions associated with Crohn's disease, and second, we sought to explore a potential molecular basis of the skin lesions in patients with CD . In this regard, we analyzed skin and GI tract biopsies from affected patients for the consensus bacterial SrRNA to determine whether direct bacterial infection was associated with either condition . Formalin-fixed, paraffin-embedded sections were studied and correlated to clinical presentation and histories from 33 patients with CD . Consensus bacterial RNA sequences were analyzed using an RT in situ PCR assay on both skin biopsy and GI biopsy material . The GI tract material included biopsies from 3 patients who had skin lesions and from 7 patients in whom there were no known skin manifestations . There were 8 cases of neutrophilic dominant dermal infiltrates, including pyoderma gangrenosum, 6 cases of granuloma annulare/necrobiosis lipoidica-like lesions, 5 cases of sterile neutrophilic folliculitis, 5 cases of panniculitis, 4 cases of vasculitis, 2 cases of psoriasis, 2 cases of lichenoid and granulomatous inflammation, and 1 case of classic metastatic CD . Intracellular bacterial 16S rRNA was detected in 8 of 10 tissues of active CD in the GI tract, of which 3 of the cases tested were from patients who also developed skin lesions at some point in their clinical course; in contrast, none of the skin biopsies had detectable bacterial RNA . The dermatopathological manifestations of CD discontiguous from the involved GI tract mucosa have in common a vascular injury syndrome, typically with a prominent extravascular neutrophilic and/or histiocytic dermal infiltrate . In addition, this study, the first to document in situ intracellular consensus bacterial SrRNA in the GI tract in CD, suggests that hematogenous dissemination of viable microbes is not associated with the cutaneous manifestations of this disease . Bacteria do, however, appear to play a role in bowel lesions of patients with CD.

Annu Rev Entomol, 2004, 49, 193 - 217
Population genetics of autocidal control and strain replacement; Gould F et al.; The concept that an insect species' genome could be altered in a manner that would result in the control of that species (i.e., autocidal control) or in the replacement of a pestiferous strain of the species with a more benign genotype was first proposed in the mid-twentieth century . A major research effort in population genetics and ecology followed and led to the development of a set of classical genetic control approaches that included use of sterile males, conditional lethal genes, translocations, compound chromosomes, and microbe-mediated infertility . Although there have been a number of major successes in application of classical genetic control, research in this area has declined in the past 20 years for technical and societal reasons . Recent advances in molecular biology and transgenesis research have renewed interest in genetically based control methods because these advances may remove some major technical problems that have constrained effective genetic manipulation of pest species . Population genetic analyses suggest that transgenic manipulations may enable development of strains that would be 10 to over 100 times more efficient than strains developed by classical methods . Some of the proposed molecular approaches to genetic control involve modifications of classical approaches such as conditional lethality, whereas others are novel . Experience from the classical era of genetic control research indicates that the population structure and population dynamics of the target population will determine which, if any, genetic control approaches would be appropriate for addressing a specific problem . As such, there continues to be a need for ongoing communication between scientists who are developing strains and those who study the native pest populations.

Int J Biochem Cell Biol, 2004 Feb, 36(2), 183 - 8
Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders; Aksoy E et al.; Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs) . TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity . Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand . A novel PKC epsilon (epsilon) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS . Thus, controlling the kinase activity of PKC epsilon might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin.

J Theor Biol, 2004 Jan 21, 226(2), 205 - 14
Evolutionary dynamics of invasion and escape; Iwasa Y et al.; Whenever life wants to invade a new habitat or escape from a lethal selection pressure, some mutations may be necessary to yield sustainable replication . We imagine situations like (i) a parasite infecting a new host, (ii) a species trying to invade a new ecological niche, (iii) cancer cells escaping from chemotherapy, (iv) viruses or microbes evading anti-microbial therapy, and also (v) the repeated attempts of combinatorial chemistry in the very beginning of life to produce self-replicating molecules . All such seemingly unrelated situations have a common structure in terms of Darwinian dynamics: a replicator with a basic reproductive ratio less than one attempts to find some mutations that allow indefinite survival . We develop a general theory, based on multitype branching processes, to describe the evolutionary dynamics of invasion and escape.

Biochim Biophys Acta, 2003 Dec 5, 1624(1-3), 54 - 9
Combination effects of complement regulatory proteins and anti-complement polymer; Yoshioka Y et al.; We previously reported the development of a "cytomedicine" that consists of cells trapped in alginate-poly-L-lysine-alginate (APA) microcapsules and agarose microbeads . The functional cells that are entrapped in semipermeable polymer are completely isolated from cellular immune system . However, the ability of cytomedicine to isolate cells from the humoral immune system, which plays an essential role in xenograft rejection, is low . Therefore, the goal of the present study was to develop a novel cytomedicine that could protect the entrapped cells from injury of the complement system . We investigated the applicability of the complement regulatory protein (CRP), Crry, to cytomedicine . Crry-transfected cells entrapped within agarose microbeads resisted injury by complement to a degree, while entrapment of Crry transfected cells within agarose microbeads containing polyvinyl sulfate (PVS), a novel cytomedical device with anti-complement activity, clearly protected against complement attack . These data indicate that the combination of a CRP and a cytomedical device with anti-complement activity is a superior device for cytomedical therapy.

Pediatr Allergy Immunol, 2003 Oct, 14(5), 371 - 7
Mold-specific immunoglobulin G antibodies in a child population; Korppi M et al.; The determination of immunoglobulin G (IgG) antibodies to molds has been used as an objective evidence of significant mold exposure . Until present, no data have been published on antibody responses to molds in healthy children living in normal housing conditions . The microbe-specific IgG antibody concentrations of 21 molds and 3 actinobacteria were determined by enzyme-linked immunosorbent assay (ELISA) in 103 1- to 6-year-old children (12.4% of the population of that age), and in 111 7- to 14-year-old school children (12.1%) . The international standard sera were available, and the IgG concentrations of the test sera could be expressed in mg/l . On average, IgG concentrations increased in relation to age until the age of 6-7 years . At school age the increase still continued but more slowly . Actinobacteria were the only exceptions; all three tested strains Sreptomyces albus, S . griseus and S . halstedii resulted in rather high concentrations until 3 years of age . If the children lived in a farm, mold-specific IgG concentrations increased at an earlier age than in other children . The results between farmers' children and other children differed significantly before school age for 20 of the 24 microbes tested, the four exceptions being the 3 actinobacteria and the mold Aspergillus versicolor . The reference values must be age related, and separate references are needed for farmers' children before school age.

Expert Opin Biol Ther, 2003 Dec, 3(8), 1279 - 89
Defensive applications of gene transfer technology in the face of bioterrorism: DNA-based vaccines and immune targeting; Ackley CJ et al.; Gene transfer involves the introduction of an engineered gene into a person's cells with the expectation that the protein expressed from the gene will produce a therapeutic benefit . Strategies based on this principle have led to the approval of > 600 clinical trials and enrollment of approximately 3500 subjects worldwide in attempts to treat diseases ranging from cancer to AIDS to cystic fibrosis . While gene therapy has met with limited success and still has many hurdles to overcome before it sees wide application, it may be useful as a defensive strategy against bioterrorism agents including infectious microbes and toxins . Although many defensive strategies are possible, immunological strategies are currently the most developed and are being actively applied to the development of strategies against several of the most virulent potential bio-weapons . While most of these strategies are not yet ready for human application, DNA-based vaccines appear to be among the most promising in the fight against bioterrorism.

Radiat Res, 2003 Dec, 160(6), 667 - 76
Assessment of proton microbeam analysis of 11B for quantitative microdistribution analysis of boronated neutron capture agents in biological tissues; Bench G et al.; The (11)B(p,alpha)(8)Be* nuclear reaction was assessed for its ability to quantitatively map the in vivo subcellular distribution of boron within gliosarcomas treated with a boronated neutron capture therapy agent . Intracranial 9L gliosarcomas were produced in Fischer 344 rats . Fourteen days later, the majority of the rats were treated with f-boronophenylalanine and killed humanely 30 or 180 min after intravenous injection . Freeze-dried tumor cryosections were imaged using the (11)B(p,alpha)(8)Be* nuclear reaction and proton microbeams obtained from the nuclear microprobe at Lawrence Livermore National Laboratory . The (11)B distributions within cells could be imaged quantitatively with spatial resolutions down to 1.5 microm, minimum detection limits of 0.8 mg/kg, and acquisition times of several hours . These capabilities offer advantages over alpha-particle track autoradiography, electron energy loss spectroscopy, and secondary ion mass spectrometry (SIMS) for quantification of (11)B in tissues . However, the spatial resolution, multi-isotope capability, and analysis times achieved with SIMS are superior to those achieved with (11)B(p,alpha)(8)Be* analysis . When accuracy in quantification is crucial, the (11)B(p,alpha)(8)Be* reaction is well suited for assessing the microdistribution of (11)B . Otherwise, SIMS may well be better suited to image the microdistribution of boron associated with neutron capture therapy agents in biological tissues.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2003 Oct, 25(5), 640 - 4
{Preparation methods of nanocavity biomaterials with recognition specificity via template imprinting of proteins}; Lu SL et al.; Nanocavity biomaterials with recognition specificity imprinted by using proteins as templates may successful serve as substitutes for antibodies, enzymes, and other native biological structures as well as cell bracket materials . It has numerous applications in biotechnology, medicine and so on . In this paper, the principle of template imprinting is introduced briefly, the specialty of template imprinting of proteins is analyzed, and the methods of template imprinting of proteins including protein entrapment, microbead surface imprinting, flat surface imprinting as well as the epitope are reviewed in details.

J Environ Sci Health B, 2003 Nov, 38(6), 683 - 95
Dissipation of the herbicide clopyralid in an allophanic soil: laboratory and field studies; Ahmad R et al.; Soil dissipation of the herbicide clopyralid (3,6-dichloropicolinic acid) was measured in laboratory incubations and in field plots under different management regimes . In laboratory studies, soil was spiked with commercial grade liquid formulation of clopyralid (Versatill, 300 g a.i . L(-1) soluble concentrate) @ 0.8 microg a.i . g(-1) dry soil and the soil water content was maintained at 60% of water holding capacity of the soil . Treatments included incubation at 10 degrees C, 20 degrees C, 30 degrees C, day/night cycles (25/15 degrees C) and sterilized soil (20 degrees C) . Furthermore, a field study was conducted at the Waikato Research Orchard near Hamilton, New Zealand starting in November 2000 to measure dissipation rates of clopyralid under differing agricultural situations . The management regimes were: permanent pasture, permanent pasture shielded from direct sunlight, bare ground, and bare ground shielded from direct sunlight . Clopyralid was sprayed in dilute solution @ 600 g a.i . ha(-1) on to field plots . Herbicide residue concentrations in soil samples taken at regular intervals after application were determined by gas chromatograph with electron capture detector . The laboratory experiments showed that dissipation rate of clopyralid was markedly faster in non-sterilized soil (20 degrees C), with a half-life (t1/2) of 7.3 d, than in sterilized soil (20 degrees C) with t1/2 of 57.8 d, demonstrating the importance of micro-organisms in the breakdown process . Higher temperatures led to more rapid dissipation of clopyralid (t1/2, 4.1 d at 30 degrees C vs 46.2 d at 10 degrees C) . Dissipation was also faster in the day/night (25/15 degrees C) treatment (t1/2, 5.4 d), which could be partly due to activation of soil microbes by temperature fluctuations . In the field experiment, decomposition of clopyralid was much slower in the shaded plots under pasture (t1/2, 71.5 d) and bare ground (t1/2, 23.9 d) than in the unshaded pasture (t1/2, 5.0 d) and bare ground plots (t1/2, 12.9 d) . These studies suggest that environmental factors such as temperature, soil water content, shading, and different management practices would have considerable influence on rate of clopyralid dissipation.

Glycobiology, 2004 Mar, 14(3), 205 - 17 Epub 2003 Nov 24.
Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures; Miller-Podraza H et al.; Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes . Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures . We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides . We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin . The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization . Binding experiments confirmed a preference of H . pylori for 3-linked sialic acid and penultimate 4-linked galactose . As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not . Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids . Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains . Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder . The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 217 - 23
The development of phenanthrene catabolism in soil amended with transformer oil; Lee PH et al.; Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants frequently associated with light non-aqueous-phase liquids (LNAPLs) in soil . Microbial degradation comprises a major loss process for PAHs in the environment . Various laboratory studies, using known degraders, have shown reduced or enhanced mineralisation of PAHs when dissolved in different LNAPLs . Effects due to the presence of LNAPLs on indigenous micro-organisms, however, are not fully understood . A pristine pasture soil was spiked with {14C}phenanthrene and transformer oil to 0, 0.01 and 0.1%, and incubated for 180 days . The catabolic potential of the soil towards phenanthrene was assessed periodically during ageing . The extent of the lag phase (prior to >5% mineralisation), maximum rates and overall extents of mineralisation observed during the course of a 14-day bioassay appeared to be dependent upon phenanthrene concentration, the presence of transformer oil, and soil-contaminant contact time . Putatively, transformer oil enhanced acclimation and facilitated the development of measurable catabolic activity towards phenanthrene in a previously uncontaminated pasture soil . Exact mechanisms for the observed enhancement, longer-term fate/degradation of the oil and residual phenanthrene, and effects of the presence of the oil on the indigenous microbes over extended time frames warrant further investigation.

J Biotechnol, 2003 Dec 5, 106(1), 1 - 13
Microbead display of proteins by cell-free expression of anchored DNA; Nord O et al.; Gene expression technologies where nucleic acid sequences remain physically linked to their corresponding gene products are important tools for selection and identification of rare variants in large protein libraries . Here, we describe a gene expression system, which combines the potential of bead-based suspension array technology (SAT) with gene expression and clonal identification . Using streptavidin-coated polystyrene micrometer-sized beads as solid supports for anchored PCR products, we have investigated conditions for cell-free expression and bioaffinity technology to provide clonal co-anchoring of corresponding gene products . Experiments showed that coupled transcription and translation of PCR product expression cassettes resulted in display of affinity-anchored proteins whose binding characteristics could be analyzed via direct and selective interaction with a fluorescently labeled target protein . Interestingly, experiments performed with differently biotinylated PCR products showed that the efficiency of display was dependent on the directionality of the expression cassette relative to the bead surface . In spiked systems, using small immunoglobulin binding proteins as models, we demonstrate efficient flow cytometric sorting of beads corresponding to the target interacting clones, verified by post-sorting analysis and clonal identification at DNA level . The use of this technology, including alternative formats, for different proteomics applications is discussed.

Scand J Immunol, 2003 Dec, 58(6), 620 - 7
Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice; Iiyama R et al.; MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes . To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice . In MyD88-/- mice, the organogenesis of PPs was not disturbed . However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice . Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly . The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks . PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide . The development of CP and IEL was also normal in 10-week-old MyD88-/- mice . These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.

Anal Chem, 2003 Aug 15, 75(16), 4161 - 7
Information coding in artificial olfaction multisensor arrays; Albert KJ et al.; High-density sensor arrays were prepared with microbead vapor sensors to explore and compare the information coded in sensor response profiles following odor stimulus . The coded information in the sensor-odor response profiles, which is used for odor discrimination purposes, was extracted from the microsensor arrays via two different approaches . In the first approach, the responses from individual microsensors were separated (decoded array) and independently processed . In the second approach, response profiles from all microsensors within the entire array, i.e., the sensor ensemble, were combined to create one response per odor stimulus (nondecoded array) . Although the amount of response data is markedly reduced in the second approach, the system shows comparable odor discrimination rates for the two signal extraction methods . The ensemble approach streamlines system resources without decreasing system performance . These signal compression approaches may simulate or parallel information coding in the mammalian olfactory system.

Blood, 2004 Apr 1, 103(7), 2677 - 82 Epub 2003 Nov 20.
New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population; Jedema I et al.; For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells . An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method . Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny . To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days . This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines.

FEBS Lett, 2003 Nov 27, 555(1), 57 - 61
Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport; DeCoursey TE; Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase . Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2- . Electron flux is electrogenic, and rapidly depolarizes the membrane potential . Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux . Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.

Curr Opin Immunol, 2003 Dec, 15(6), 614 - 9
Do microbes influence the pathogenesis of allergic diseases? Building the case for Toll-like receptor ligands; Horner AA et al.; The prevalence and severity of allergic diseases and other diseases of immune dysregulation are increasing in industrialized countries . One explanation for these trends is that decreased exposure to microbes, due to modern public health practices, has resulted in the loss of a main source of immune provocation, and a consequent increase in pathogenic immune responses and their associated diseases . It is now clear that molecular interactions between immunocytes and microbes are mediated largely by Toll-like receptors (TLRs) on host cells and a diversity of ligands produced by viruses, bacteria and fungi . Physiological exposures to TLR ligands are also likely to have an important yet complex role in host immune homeostasis and predisposition towards atopy.

Neoplasma, 2003, 50(5), 350 - 6
Hairy cell leukemia: early immunophenotypical detection and quantitative analysis by flow cytometry; Babusikova O et al.; The abnormal coexpression of the so-called 'HCL-restricted' markers (CD22+CD11c, CD25 and CD103) identified on monotypic, slightly large B-lymphocytes in the large cell-gate of dot-plots has previously been shown to be highly characteristic of hairy cell leukemia (HCL) . The main aim of our present study was to determine if patterns with low levels of neoplastic cells in bone marrow (BM) or peripheral blood (PB) are of a value the early diagnosis and/or detection of minimal residual disease (MRD) in HCL . Next we wished to determine if quantitative immunophenotyping given by molecules of equivalent soluble fluoresceine (MESF) could help to distinguish pathologic B-lymphocytic pool from that of normal residual B-cells also in patients with low numbers of HCL cells . The abnormal immunophenotypes were studied in 174 specimens from 19 patients with suspect HCL or during follow-up of already treated patients . For evaluation of marker density fluorescent calibration microbeads were used . In 12 HCL patients (67%) permanent complete remission was observed after treatment . In 6 patients (33%) transient MRD+ phenotype was identified but the clinical manifestation of relapse was followed till now in only three patients . One patient was phenotyped just only at diagnosis . The pathological cells in low levels were found in 5 patients at diagnosis (in the range from 2 to 12%) and in patients with MRD+ phenotype they were recognized repeatedly in the range from 2 to 8% . Furthermore, we observed in hairy cells significantly higher values of molecule numbers of some B-cell markers, comparing to that of residual B-cells in nonleukemic lymphocyte gate of the same sample . We found profound and persistent CD4+ lymphopenia in all but one studied patients after CdA treatment . Conclusions: Flow cytometric immunophenotyping of PB and BM is highly sensitive and specific method and is capable to detect low levels of malignant cells in HCL . Quantitative analysis of MESF values of pathological B-cells comparing to normal residual B-cells seems to be another new marker of HCL in common, which is reliable detecting also small cell numbers in examined sample . A long-term decline of CD4+ T-cells correlated with the relatively low incidence of clinical progression of HCL.

Neuroscience, 2003, 122(3), 647 - 57
Interplay between brain-derived neurotrophic factor and signal transduction modulators in the regulation of the effects of exercise on synaptic-plasticity; Vaynman S et al.; This study was designed to identify molecular mechanisms by which exercise affects synaptic-plasticity in the hippocampus, a brain area whose function, learning and memory, depends on this capability . We have focused on the central role that brain-derived neurotrophic factor (BDNF) may play in mediating the effects of exercise on synaptic-plasticity . In fact, this impact of exercise is exemplified by our finding that BDNF regulates the mRNA levels of two end products important for neural function, i.e . cAMP-response-element binding (CREB) protein and synapsin I . CREB and synapsin I have the ability to modify neuronal function by regulating gene-transcription and affecting synaptic transmission, respectively . Furthermore, we show that BDNF is capable of concurrently increasing the mRNA levels of both itself and its tyrosine kinaseB (TrkB) receptor, suggesting that exercise may employ a feedback loop to augment the effects of BDNF on synaptic-plasticity . The use of a novel microbead injection method in our blocking experiments and Taqman reverse transcription polymerase reaction (RT-PCR) for RNA quantification, have enabled us to evaluate the contribution of different pathways to the exercise-induced increases in the mRNA levels of BDNF, TrkB, CREB, and synapsin I . We found that although BDNF mediates exercise-induced hippocampal plasticity, additional molecules, i.e . the N-methyl-D-aspartate receptor, calcium/calmodulin protein kinase II and the mitogen-activated protein kinase cascade, modulate its effects . Since these molecules have a well-described association to BDNF action, our results illustrate a basic mechanism through which exercise may promote synaptic-plasticity in the adult brain.

Reprod Nutr Dev, 2003 May-Jun, 43(3), 271 - 84
II . Quantitative aspects of phosphorus absorption in ruminants; Bravo D et al.; Phosphorus absorption in ruminants was analysed from a database described in a previous article . For common values of ingested phosphorus (2.5-5.0 g x kg(-1) of DM), 0.73 of dietary phosphorus is absorbed . The remaining variability is probably due to phosphorus quality . Phosphorus absorbed from silage, cereal, cereal by-products and hay differs greatly . The current true absorption coefficient used to calculate daily phosphorus supply is a constant value in the current systems and often it underestimates the true absorption resulting in an excess of phosphorus being supplied in the diets . Adjusting the true absorption coefficient values requires better characterisation of the phosphorus supplied by each feedstuff . Dietary influences (phytate phosphorus, crude fibre, etc.) were investigated but trials assessing the ration effect on phosphorus absorption are lacking . Since rumen microbes have specific phosphorus requirements, particularly for cell-wall degradation, the feedstuffphosphorus availability for the rumen ecosystem is discussed.

Scand J Infect Dis, 2003, 35(9), 628 - 31
Bacterial protein toxins and inflammation; Soderblom T et al.; Although human mucosal linings are continuously exposed to microbes, the microbes rarely induce disease . This is because mucosal surfaces are protected by a first line of defence termed the innate immunity system . Inflammatory processes are activated as a consequence of a complex interplay between microbes and host target cells . Although inflammation is essential for clearing out infectious agents, it can also be harmful to the host and is therefore subjected to tight control at multiple levels . It was recently discovered that the bacterial protein toxin alpha-haemolysin (HlyA), secreted by uropathogenic Escherichia coli, induces constant, low-frequency Ca2+ oscillations in renal epithelial cells . Ca2+ oscillation occurs at a characteristic periodicity of 12 min, and affects gene expression in target epithelial cells . Specifically, the proinflammatory cytokine interleukin-6 (IL-6) and chemokine IL-8 were induced by HlyA-induced Ca2+ oscillations . A few additional bacterial protein toxins have been reported to induce Ca2+ oscillations in target epithelial cells, although their effects are poorly understood . However, the pioneering work on HlyA demonstrates a novel feature of bacterial protein toxins on host target cells: as inducers of second messenger responses which fine-tune gene expression in target epithelial cells.

Biofouling, 2003 Feb, 19(1), 65 - 76
Microbially influenced corrosion as a model system for the study of metal microbe interactions: a unifying electron transfer hypothesis; Hamilton WA; The general term biomineralisation refers to biologically induced mineralisation in which an organism modifies its local microenvironment creating conditions such that there is chemical precipitation of mineral phases extracellularly . Most usually this results from an oxidation or reduction carried out by some microbial species, with the formation of a recognised biomineralised product . These reactions play a major role in microbial physiology and ecology, and are of central importance to such engineering consequences as microbial mining and microbially influenced corrosion . This paper will examine metal microbe interactions, both in naturally occurring microbial ecosystems and in two particular cases of biocorrosion, with the objective of putting forward a unifying hypothesis relevant to the understanding of each of these apparently disparate processes.

Anal Chem, 2003 Nov 15, 75(22), 6327 - 33
Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers; Lapierre MA et al.; The detection of specific DNA sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens . A new assay developed for this purpose is described that harnesses a sensitive electrocatalytic process to monitor DNA hybridization . Two sequences belonging to the pathogenic microbe Helicobacter pylori are used to demonstrate the versatility and specificity of the assay: one that codes for an unique H . pylori protein and one that represents a small portion of the 23S rRNA from this organism . Both sequences can be monitored into the nanomolar concentration range . Target sequences introduced to the electrode surface as synthetic oligonucleotides, PCR products, and RNA transcripts are all detected with high specificity . In addition to reporting the presence of pathogen-related sequences, this assay can accurately resolve single-base changes in target sequences . An A2143C substitution within the H . pylori rRNA that confers antibiotic resistance significantly attenuates hybridization to an immobilized probe corresponding to the WT sequence . The single-base mismatch introduced by this mutation slows the kinetics of hybridization and permits discrimination of the two sequences when short hybridization times are employed . The remarkable sensitivity of this label-free assay to small sequence changes may provide the basis of a new method for the detection and genotyping of infectious bacteria using electrochemical methods.

Microbes Infect, 2003 Nov, 5(14), 1307 - 15
Oxidative killing of microbes by neutrophils; Roos D et al.; Neutrophils and other phagocytic leukocytes contain a phagocyte NADPH oxidase enzyme that generates superoxide after cell activation . Reactive oxygen species derived from superoxide, together with proteases liberated from the granules, are used to kill ingested microbes . Dysfunction of the phagocyte NADPH oxidase results in chronic granulomatous disease, with life-threatening infections.

Int J Mol Med, 2003 Dec, 12(6), 969 - 75
Rare expression of target antigens for immunotherapy on disseminated tumor cells in breast cancer patients without overt metastases; Kasimir-Bauer S et al.; Occult disseminated tumor cells are the major cause of relapse in patients with primary operable breast cancer but detection and characterization of these few cells is difficult . Applying immunohistochemistry, an immunomagnetic enrichment technique (IET) and immunocytochemistry (IC), we studied 58 breast cancer patients without overt metastases for the frequency of cytokeratin-positive (CK+) bone marrow (BM) cells coexpressing the epithelial adhesion molecule 17-1A (EpCAM) and c-erbB-2 and analyzed the primary tumor for these antigens as a strategy for additional immunotherapy . The primary tumors were analyzed for the target antigens by a pathologist . Dissemination of CK+ cells was studied in 4-6 x 10(6) BM cells by IC alone . For characterization of CK+ cells, 10-15 x 10(6) BM cells were incubated with microbeads coupled to antibodies detecting the target antigens, labelled cells were separated on selection columns and the positively (BM cells carrying the target antigen) and negatively (BM cells without target antigen) selected fractions were stained for CK+ cells . The effectiveness of these methods was confirmed in cell culture models . 17-1A was detected in all primary tumors and c-erbB-2 overexpression (2+, 3+) was found in 25/58 tissue samples . In total, analyzing 15-20 x 10(6) BM cells in each patient, the detection rate for CK+ cells in the BM was 69% (40/58 patients) . Interestingly, analysis of the positive and negative enrichment fractions showed that the 17-1A antigen was coexpressed on CK+ cells in only 6 patients and c-erbB-2/CK+ cells were found in only one patient . Although 17-1A and c-erbB-2 were frequently detected in the primary tumor, these antigens were rarely expressed on CK+ BM cells . Whether the applied IET is not able to detect low amounts of these target antigens has to be clarified . Nevertheless, applying cell-cycle independent protocols in clinical trials requires careful elucidation of those patients who might benefit from these therapies.

Hepatobiliary Pancreat Dis Int, 2002 Aug, 1(3), 354 - 67
D-galactosamine based canine acute liver failure model; Patzer JF 2nd et al.; BACKGROUND: Appropriate preclinical evaluation of a bioartificial liver assist device (BAL) demands a large animal model, as presented here, that demonstrates many of the clinical features of acute liver failure and that is suitable for clinical qualitative and quantitative evaluation of the BAL . A lethal canine liver failure model of acute hepatic failure that removes many of the artifacts evidenced in prior canine models is presented . METHODS: Six male hounds, 24-30 kg, under isoflurane anesthesia, were administered 1.5 g/kg D-galactosamine intravenously . Canine supportive care followed a well-defined management protocol that was guided by electrolyte and invasive monitoring consisting of arterial pressure, central venous pressure, extradural intracranial pressure (ICP), pulmonary artery pressure, and end-tidal CO2 . The animals were treated until death-equivalent, defined as inability to sustain systolic blood pressure >80 mmHg for 20 minutes despite maximal fluids and 20 microg/kg/min dopamine infusion . RESULTS: The mean survival time was 43.7+/-4.6 hours (mean+/-SE) . All animals showed evidence of progressive liver failure characterized by increasing liver enzymes (aspartate transaminase from 26 to 5977 IU/L; alanine transaminase from 32 to 9740 IU/L), bilirubin (0.25 to 1.30 mg/dl), ammonia (19.8 to 85.3 micromol/L), and coagulopathy (prothrombin time from 8.7 to 46 s) . Increased lability and elevations in intracranial pressures were observed . All animals were refractory to maintenance of cerebral perfusion pressure even with only moderately elevated intracranial pressure . Severe neurologic obtundation, seen in 2 of 6 animals, was associated with elevations of ICP above 50 mmHg . Post-mortem liver histology showed evidence of massive hepatic necrosis . Postmortem blood and ascites microbial growth was consistent with possible translocation of intestinal microbes . CONCLUSIONS: The improved lethal canine liver failure model presented here reproduces many of the clinical features of acute liver failure . The model may prove useful for qualitative and quantitative evaluation of BALs.

Science, 2003 Nov 7, 302(5647), 1041 - 3
Control of effector CD8+ T cell function by the transcription factor Eomesodermin; Pearce EL et al.; Activated CD8+ T cells play a critical role in host defense against viruses, intracellular microbes, and tumors . It is not clear if a key regulatory transcription factor unites the effector functions of CD8+ T cells . We now show that Eomesodermin (Eomes), a paralogue of T-bet, is induced in effector CD8+ T cells in vitro and in vivo . Ectopic expression of Eomes was sufficient to invoke attributes of effector CD8+ T cells, including interferon-gamma (IFN-gamma), perforin, and granzyme B . Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of CD8+ T cells . We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.

Am J Physiol Heart Circ Physiol, 2004 Mar, 286(3), H918 - 25 Epub 2003 Nov 06.
Spatial and temporal control of angiogenesis and arterialization using focal applications of VEGF164 and Ang-1; Peirce SM et al.; Microvascular networks undergo patterning changes that determine and reflect functional adaptations during tissue remodeling . Alterations in network architectures are a result of complex and integrated signaling events . To understand how two growth factor signals interact to stimulate angiogenesis and arterialization, we engineered spatially directed microvascular pattern changes in vivo by using combinations of focally delivered exogenous growth factors . We implanted microdelivery beads containing recombinant vascular endothelial growth factor-164 (VEGF(164)) and recombinant angiopoietin-1* (Ang-1*) into the dorsal subcutaneous tissue of fully anesthetized male Fischer 344 rats implanted with backpack window chambers, and we quantified vascular patterning changes by using intravital microscopy, a combination of architectural metrics, and immunohistochemistry . Focal delivery of VEGF(164) caused spatially directed increases in both the total number and the density of vessels with diameters <25 microm 7 days after microbead implantation . Increases were maintained out to 14 days but were reduced to control values by day 21 . The addition of Ang-1* on day 7 maintained these increases out to day 21, induced vessel order ratios comparable to control levels, and was accompanied by increases in the length density of smooth muscle alpha-actin-positive vessels . We achieved spatial control of patterning changes in vivo by using multisignal stimulation via focal delivery of exogenous growth factor combinations and conclude that Ang-1* administered subsequent to VEGF(164) stimulation induces vascular growth while maintaining a network pattern consistent with native patterns that persist in the presence of vehicle control stimulation.

Prog Nucleic Acid Res Mol Biol, 2003, 75, 217 - 92
Dancing with complement C4 and the RP-C4-CYP21-TNX (RCCX) modules of the major histocompatibility complex; Yu CY et al.; The number of the complement component C4 genes varies from 2 to 8 in a diploid genome among different human individuals . Three quarters of the C4 genes in Caucasian populations have the endogenous retrovirus, HERV-K(C4), in the ninth intron . The remainder does not . The C4 serum proteins are highly polymorphic and their concentrations vary from 100 to approximately 1000 microg/ml . There are two distinct classes of C4 protein, C4A and C4B, which have diversified to fulfill (a) the opsonization/immunoclearance purposes and (b) the well-known complement function in the killing of microbes by lysis and neutralization, respectively . Many infectious and autoimmune diseases are associated with complete or partial deficiency of C4A and/or C4B . The adverse effects of high C4 gene dosages, however, are just emerging, as the concepts of human C4 genetics are revised and accurate techniques are applied to distinguish partial deficiencies from differential expression caused by unequal C4A and C4B gene dosages and gene sizes . This review attempts to dissect the sophisticated genetics of complement C4A and C4B . The emphases are on the qualitative and quantitative diversities of C4 genotypes and phenotypes . The many allotypic variants and the processed products of human and mouse C4 proteins are described . The modular variation of C4 genes together with the serine/threonine nuclear kinase gene RP, the steroid 21-hydroxylase CYP21, and extracellular matrix protein TNX (RCCX modules) are investigated for the effects on homogenization of C4 protein polymorphisms, and on the unequal genetic crossovers that knocked out the functions of CYP21 and/or TNX . Furthermore, the influence of the endogenous retrovirus HERV-K(C4) on C4 gene expression and the dispersal of HERV-K(C4) family members in the human genome are discussed.

Appl Environ Microbiol, 2003 Nov, 69(11), 6825 - 32
Comparative genomics of insect-symbiotic bacteria: influence of host environment on microbial genome composition; Rio RV et al.; Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism . Using macroarrays developed for a close relative, Escherichia coli, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations: Sodalis glossinidius with the tsetse fly (Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with the rice weevil (Coleoptera, Sitophilus oryzae) . Functional biologies within their hosts currently reflect different forms of symbiotic associations . Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil . Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport . The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for Sodalis and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE . Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state . It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.

Appl Environ Microbiol, 2003 Nov, 69(11), 6550 - 9
Members of a readily enriched beta-proteobacterial clade are common in surface waters of a humic lake; Burkert U et al.; Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment . We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats . Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs . These changes were compared to community shifts induced during short-term enrichment experiments . Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation . The dilutions were incubated for a period of 2 weeks . The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes . beta-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments . In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations . In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column . Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment . Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments . We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.

Zh Obshch Biol, 2003 Sep-Oct, 64(5), 389 - 402
{Annual cyclic changes and self-organization processes in the marine sea-bottom organisms}; Burkovskii IV et al.; The dynamics of marine microbenthos species structure shows clear annual cycle . From late winter till early autumn community changes towards increasing complexity and orderliness . The sharp increase in available energy (light and heat) in the beginning of the winter acts as trigger of these processes . The further development of the community is connected with system fluctuations arising as a result of interactions or organisms between each other and their environment (feeding, competition, predation) . In the end of summer these processes are terminated by the state of high species diversity, maximum species coordination, expansion of inhabited zone within the sediments, and the distinct segregation of space and nutrient resources among species (i.e., achievement of ecological complementarity) . Decrease in light and temperature causes the reverse process--weakening of organism coordination and significant simplification of the community structure . In general, these changes correspond to the theory of self-organization in nonequilibrium systems (Prigogine, Stengers, 2001) . The most important distinction of observed processes from the classical self-organization is their cyclic dynamics, i.e . the annual return of community to its most simple state at the end of winter.

FASEB J, 2004 Jan, 18(1), 203 - 5 Epub 2003 Nov 03.
The LPS receptor (CD14) links innate immunity with Alzheimer's disease; Fassbender K et al.; To rapidly respond to invading microorganisms, humans call on their innate immune system . This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens . Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia . We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide . Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity . The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings . These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Am J Vet Res, 2003 Oct, 64(10), 1278 - 82
Pharmacokinetics of ceftiofur sodium after intramuscular or subcutaneous administration in green iguanas (Iguana iguana); Benson KG et al.; OBJECTIVE: To determine the pharmacokinetics of ceftiofur sodium after IM and SC administration in green iguanas . ANIMALS: 6 male and 4 female adult green iguanas . PROCEDURE: In a crossover design, 5 iguanas received a single dose of ceftiofur sodium (5 mg/kg) IM, and 5 iguanas received the same dose SC . Blood samples were taken at 0, 20, and 40 minutes and 1, 2, 4, 8, 24, 48, and 72 hours after administration . After a 10-week washout period, each iguana was given the same dose via the reciprocal administration route, and blood was collected in the same fashion . Ceftiofur free-acid equivalents were measured via high-performance liquid chromatography . RESULTS: The first phase intercepts were significantly different between the 2 administration routes . Mean maximum plasma concentration was significantly higher with the IM (28.6 +/- 8.0 microg/mL) than the SC (18.6 +/- 8.3 microg/mL) administration route . There were no significant differences between terminal half-lives (harmonic mean via IM route, 15.7 +/- 4.7 hours; harmonic mean via SC route, 19.7 +/- 6.7 hours) and mean areas under the curve measured to the last time point (IM route, 11,722 +/- 7,907 microg x h/mL; SC route, 12,143 +/- 9,633 microg x h/mL) . Ceftiofur free-acid equivalent concentrations were maintained > or = 2 microg/mL for > 24 hours via both routes . CONCLUSIONS AND CLINICAL RELEVANCE: A suggested dosing schedule for ceftiofur sodium in green iguanas for microbes susceptible at > 2 microg/mL would be 5 mg/kg, IM or SC, every 24 hours.

J Med Entomol, 2003 Sep, 40(5), 698 - 705
Sindbis virus-associated pathology in Aedes albopictus (Diptera: Culicidae); Bowers DF et al.; Virus dissemination and associated pathology were examined in Aedes albopictus after intrathoracic inoculation of Sindbis virus (SIN), the prototypic Alphavirus . At 10 days postinfection, virus RNA was detected in all three-body segments of the insect . Colocalization of virus antigen with structural pathology was observed in mosquito salivary glands and midgut-associated visceral muscles, representing yet another example of arbovirus-associated pathology in a mosquito host . SIN antigen and gross pathology were detected in lateral lobes, but not the medial lobe of salivary glands, whereas virus antigen, vacuolated cytoplasm, and myofilament misalignment were detected in the visceral muscles at the midgut exterior surface . Early in the midgut infection, virus antigen was localized in small foci on the organ surface that progressed to a grate work-like banding pattern that eventually cleared . Both the salivary glands and the midgut are essential to insect survival and reproduction . Additionally, these organs provide a pathway for virus transmission in nature . Although SIN infection may not shorten the mosquito life span, persistent coexistence could permit survival of both host and microbe as well as contribute to alterations in insect behavior.

Riv Biol, 2003 May-Aug, 96(2), 207 - 23
The ecological risks of transgenic plants; Giovannetti M; Biotechnologies have been utilized "ante litteram" for thousands of years to produce food and drink and genetic engineering techniques have been widely applied to produce many compounds for human use, from insulin to other medicines . The debate on genetically modified (GM) organisms broke out all over the world only when GM crops were released into the field . Plant ecologists, microbiologists and population geneticists carried out experiments aimed at evaluating the environmental impact of GM crops . The most significant findings concern: the spread of transgenes through GM pollen diffusion and its environmental impact after hybridisation with closely related wild species or subspecies; horizontal gene transfer from transgenic plants to soil microbes; the impact of insecticide proteins released into the soil by transformed plants on non-target microbial soil communities . Recent developments in genetic engineering produced a technology, dubbed "Terminator", which protects patented genes introduced in transgenic plants by killing the seeds in the second generation . This genetic construct, which interferes so heavily with fundamental life processes, is considered dangerous and should be ex-ante evaluated taking into account the data on "unexpected events", as here discussed, instead of relying on the "safe until proven otherwise" claim . Awareness that scientists, biotechnologists and genetic engineers cannot answer the fundamental question "how likely is that transgenes will be transferred from cultivated plants into the natural environment?" should foster long-term studies on the ecological risks and benefits of transgenic crops.

Arch Microbiol, 2003 Dec, 180(6), 455 - 64 Epub 2003 Oct 31.
Identification of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin synthase required for coenzyme F(420) biosynthesis; Graham DE et al.; The hydride carrier coenzyme F(420) contains the unusual chromophore 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) . Microbes that generate F(420) produce this FO moiety using a pyrimidine intermediate from riboflavin biosynthesis and the 4-hydroxyphenylpyruvate precursor of tyrosine . The fbiC gene, cloned from Mycobacterium smegmatis, encodes the bifunctional FO synthase . Expression of this protein in Escherichia coli caused the host cells to produce FO during growth, and activated cell-free extracts catalyze FO biosynthesis in vitro . FO synthase in the methanogenic euryarchaeon Methanocaldococcus jannaschii comprises two proteins encoded by cofG (MJ0446) and cofH (MJ1431) . Both subunits were required for FO biosynthesis in vivo and in vitro . Cyanobacterial genomes encode homologs of both genes, which are used to produce the coenzyme for FO-dependent DNA photolyases . A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages . Ancestors of the cyanobacteria and actinomycetes must have acquired the two genes, which subsequently fused in actinomycetes . Both CofG and CofH have putative radical S-adenosylmethionine binding motifs, and pre-incubation with S-adenosylmethionine, Fe(2+), sulfide, and dithionite stimulates FO production . Therefore a radical reaction mechanism is proposed for the biosynthesis of FO.

Arterioscler Thromb Vasc Biol, 2004 Feb, 24(2), 230 - 9 Epub 2003 Oct 30.
Decoding transcriptional programs regulated by PPARs and LXRs in the macrophage: effects on lipid homeostasis, inflammation, and atherosclerosis; Ricote M et al.; Macrophages play essential roles in immunity and homeostasis . As professional scavengers, macrophages phagocytose microbes and apoptotic and necrotic cells and take up modified lipoprotein particles . These functions require tightly regulated mechanisms for the processing and disposal of cellular lipids . Under pathological conditions, arterial wall macrophages become foam cells by accumulating large amounts of cholesterol, contributing to the development of atherosclerosis . Peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs) are members of the nuclear receptor superfamily of transcription factors that have emerged as key regulators of macrophage homeostasis . PPARs and LXRs control transcriptional programs involved in processes of lipid uptake and efflux, lipogenesis, and lipoprotein metabolism . In addition, PPARs and LXRs negatively regulate transcriptional programs involved in the development of inflammatory responses . This review summarizes recent efforts to decode the differential and overlapping roles of PPARs and LXRs in the context of macrophage lipid homeostasis and the control of inflammation.

Arterioscler Thromb Vasc Biol, 2003 Dec, 23(12), 2209 - 14 Epub 2003 Oct 30.
Evidence for protein kinase C-mediated activation of Rho-kinase in a porcine model of coronary artery spasm; Kandabashi T et al.; OBJECTIVE: We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model . However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm . METHODS AND RESULTS: A segment of left porcine coronary artery was chronically treated with IL-1beta-bound microbeads in vivo . Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1beta-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor . Guanosine 5'-{gamma-thio}triphosphate (GTPgammaS), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca2+ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C . The GTPgammaS-induced Ca2+ sensitization was greater in the spastic segment than in the control segment . Western blot analysis revealed that only PKCdelta isoform was activated during the hypercontraction . CONCLUSIONS: These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKCdelta may be involved . Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.

Environ Int, 2004 Jan, 29(7), 907 - 21
Estimation of septic tank setback distances based on transport of E . coli and F-RNA phages; Pang L et al.; Setback distances between septic tank systems and the shorelines of Lake Okareka, New Zealand were determined from model simulations for a worst-case scenario, using the highest hydraulic conductivity and gradient measured in the field, removal rates of the microbial indicators (Escherichia coli and F-RNA phages) determined from a column experiment, and maximum values of the design criteria for the disposal system, and assuming an absence of an unsaturated zone, a continuous discharge of the raw effluent from a failed or non-complying treatment system (both indicators at concentrations of 1x10(7) counts/100 ml) into the groundwater and no sorption of pathogens in the aquifer . Modelling results suggest that the minimal setback distances were 16 m to satisfy the New Zealand Recreational Water Quality Guidelines for E . coli <126 per 100 ml (Ministry for the Environment, 1999) and 48 m to meet the Drinking-Water Standards for New Zealand 2000 for enteric virus <1 per 100 l (Ministry of Health, 2000) . These distances may be applicable for other lakeshores in pumice sand aquifers with groundwater velocities <7 m/day . Findings of laboratory column and batch experiments provided an insight into the microbial attenuation and transport processes in pumice sand aquifers . Bacterial removal was predominately through filtration (87-88%) and partially by die-off (12-13%), while viral removal was by both die-off (45%) and filtration (55%) . In addition, microbial die-off in groundwater without aquifer material (i.e., free microbes) was much lower than die-off in groundwater with aquifer material (i.e., sorbed microbes) and contributed only 2-6% to the total removal . This implies that the setback distances estimated from die-off rates for the free microbes, determined in the laboratory without considering aquifer media and other removal processes, which are often reported in the literature, could be larger than necessary.

Am Nat, 2003 Oct, 162(4 Suppl), S51 - 62
Multispecies microbial mutualisms on coral reefs: the host as a habitat; Knowlton N et al.; Reef-building corals associate with a diverse array of eukaryotic and noneukaryotic microbes . Best known are dinoflagellates in the genus Symbiodinium ("zooxanthellae"), which are photosynthetic symbionts found in all reef-building corals . Once considered a single species, they are now recognized as several large, genetically diverse groups that often co-occur within a single host species or colony . Variation among Symbiodinium in host identities, tolerance to stress, and ability to colonize hosts has been documented, but there is little information on the ecology of zooxanthellar free-living stages and how different zooxanthellae perform as partners . Other microbial associates of reef corals are much less well known, but studies indicate that individual coral colonies host diverse assemblages of bacteria, some of which seem to have species-specific associations . This diversity of microbial associates has important evolutionary and ecological implications . Most mutualisms evolve as balanced reciprocations that allow partners to detect cheaters, particularly when partners are potentially diverse and can be transmitted horizontally . Thus, environmental stresses that incapacitate the ability of partners to reciprocate can destabilize associations by eliciting rejection by their hosts . Coral bleaching (the loss of zooxanthellae) and coral diseases, both increasing over the last several decades, may be examples of stress-related mutualistic instability.

J Biol Chem, 2004 Jan 30, 279(5), 3218 - 27 Epub 2003 Oct 28.
Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect; Lee MH et al.; The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan . A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system . To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column . Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster . To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells . Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan . By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose . Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+) . These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.

Biophys J, 2003 Nov, 85(5), 3336 - 49
A three-dimensional viscoelastic model for cell deformation with experimental verification; Karcher H et al.; A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry . The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate . Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry . Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work . The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing . Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending <10 microm from the bead . Modulation of cell height has little effect on the results, provided the monolayer is >5 micro m thick . NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.

J Environ Manage, 2003 Nov, 69(3), 249 - 59
Transport of gellan gum microbeads through sand: an experimental evaluation for encapsulated cell bioaugmentation; Moslemy P et al.; Transport of 10-40 microm gellan gum microbeads was studied in horizontal sand columns to evaluate the feasibility of using gel-encapsulated bacteria for bioaugmentation of contaminated aquifers . Three 5.2 x 110 cm columns were packed with sand (column A: 0.5-2 mm, column B: 0.25-2 mm, and column C: 0.125-2 mm) . Microbeads in artificial groundwater were injected at 0.5 l h(-1) during intermittent 12-h periods . Breakthrough of microbeads increased with injection time, varying as a descending function of travel distance . After 72 h of injection, about 75% of injected microbeads were dispersed across a 5-110 cm distance from the inlet in column A, compared to 78% across a 5-50 cm in column B, and 76% across a 5-20 cm in column C . The wider dispersion of microbeads across the length of column A, compared to those observed in columns B and C, suggests a higher potential for the formation of a uniform bioactive zone of encapsulated cells across a sandy aquifer with such grain size distribution and hydrodynamic properties.

Radiat Prot Dosimetry, 2003, 104(4), 347 - 55
A review of the bystander effect and its implications for low-dose exposure; Prise KM et al.; Current models for the interaction between ionising radiation and living cells or tissues are based on direct genetic damage produced by energy deposition in cellular DNA . An important observation which has questioned this basic assumption is the radiation-induced bystander response, in which cells which have not been directly targeted respond if their neighbours have been exposed . This response predominates at low doses of relevance to radiation risk analysis (<0.2 Gy) and therefore needs to be fully characterised . The development of microbeams, which allow individual cells within populations to be targeted with precise doses of radiation, has provided a useful tool for quantifying this response . The authors' studies have targeted individual human and mouse cells with counted protons and helium ions and monitored neighbouring cells for the production of bystander responses . Bystander responses have been measured after exposures as low as a single proton or helium ion delivered to an individual cell . An important aspect is that these responses saturate with increasing dose to the single target cell, thus the relative roles of direct and indirect (non-targeted) responses change with dose . Studies with multicellular, tissue-based models are providing evidence that bystander responses may have a complex phenotype involving multiple pathways and the overall response may be a balance between multiple signalling processes and responses to radiation exposure . Current models for radiation risk assume a linear non-threshold response and have generally been extrapolated from high-dose exposures . The involvement of competing processes at low doses may have important consequences for understanding the effects of low-dose exposure.

Biotechniques, 2003 Oct, 35(4), 734 - 6, 738-40
Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA; Mizukami A et al.; Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts . The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution . We developed a new method of transforming yeast using chromosome-sized DNA . The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA . Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA . The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.

Eur J Immunol, 2003 Nov, 33(11), 2953 - 63
Control of inflammatory diseases by pathogens: lipids and the immune system; van der Kleij D et al.; Inflammatory diseases such as asthma and diabetes are rising in industrialized countries and the modern lifestyle that is associated with lower exposure to microbes has been held responsible for the increasing prevalence of these diseases . Several studies have shown an inverse association between pathogen-exposure and allergy or autoimmunity . The mechanisms behind such protective effects have been investigated at the epidemiological, cellular and molecular level and have provided data on the ability of lipids either derived directly from pathogens or up-regulated as a result of an infection to down-regulate immune responses and thereby control inflammatory diseases . In this mini-review, recent findings and new concepts relating to the immunosuppressive effects of endogenous lipids and those encountered upon exposure to bacteria, protozoa and particularly helminths are discussed . The overview focuses on the modulation of interactions between the antigen-presenting compartment and T cells to start an anti-inflammatory "program" with potential to regulate disease processes.

Astrobiology, 2003 Summer, 3(2), 407 - 14
Biological potential of Martian hydrothermal systems; Varnes ES et al.; A source of energy to power metabolism may be a limiting factor in the abundance and spatial distribution of past or extant life on Mars . Although a global average of chemical energy available for microbial metabolism and biomass production on Mars has been estimated previously, issues of how the energy is distributed and which particular environments have the greatest potential to support life remain unresolved . We address these issues using geochemical models to evaluate the amounts of chemical energy available in one potential biological environment, Martian hydrothermal systems . In these models, host rock compositions are based upon the compositions of Martian meteorites, which are reacted at high temperature with one of three groundwater compositions . For each model, the values for Gibbs energy of reactions that are important for terrestrial chemosynthetic organisms and likely representative for putative Martian microbes are calculated . Our results indicate that substantial amounts of chemical energy may be available in these systems, depending most sensitively upon the composition of the host rock . From the standpoint of sources of metabolic energy, it is likely that suitable environments exist to support Martian life.

J Speech Lang Hear Res, 2003 Oct, 46(5), 1247 - 61
Interarticulator coordination in dysarthria: an X-ray microbeam study; Weismer G et al.; Articulatory discoordination is often said to be an important feature of the speech production disorder in dysarthria, but little experimental work has been done to identify and specify the coordination difficulties . The present study evaluated the coordination of labial and lingual gestures for /u/ production in persons with Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and in control participants . Both tongue backing/raising and reduction of the area enclosed by the lips can produce the characteristic low F2 of /u/ . The timing of these articulatory gestures with respect to the acoustic target of a low F2 was inferred from X-ray microbeam data . Pellet motions of the tongue dorsum and lips revealed the timing of the lingual and labial gestures to be strongly linked together (synchronized), predictive of the temporal location of the lowest F2 within the vocalic nucleus, and scaled proportionately to the overall vowel duration in control participants . Somewhat surprisingly, essentially the same findings were obtained in the speakers with dysarthria . These relationships were noisier among the speakers with dysarthria, but the global synchronization patterns applied to all 3 groups . Further analyses revealed the synchronization to be less well defined and more variable across speakers with ALS, as compared to speakers with PD and the controls . Results are discussed relative to concepts of coordination in dysarthria.

Infect Immun, 2003 Nov, 71(11), 6543 - 52
Forssman synthetase expression results in diminished shiga toxin susceptibility: a role for glycolipids in determining host-microbe interactions; Elliott SP et al.; Forssman glycolipid (FG), the product of Forssman synthetase (FS), is widely expressed among nonprimate mammalian species . Here, we describe a molecular and genetic relationship between FG expression and Shiga toxin (Stx) susceptibility . We have isolated the FS cDNA from human, canine, and murine cells . Whereas the murine and canine FS genes express a functional enzyme, the human FS cDNA was found to express a protein that lacks FS activity, despite a high degree of sequence identity with the enzymatically active murine and canine FS genes . In order to examine the relationship between FG expression and Stx susceptibility, Vero cells were transfected with the three FS orthologues or a vector control . Complementation with the human FS cDNA had no effect on Stx susceptibility, whereas stable expression of the canine and murine FS resulted in markedly decreased susceptibility to toxin . Among individual cells, an inverse correlation between FG expression and Stx binding was demonstrated . Moreover, only strongly FG-reactive cells were capable of growing in the presence of Stx . These cells were found to have high levels of FG expression and a correspondingly diminished GbO(3) content . We conclude that expression of a functionally active FS modifies Stx receptor glycolipids to FG and results in markedly decreased susceptibility to toxin . We speculate that inactivation of the FS gene during primate evolution may account, at least in part, for the marked susceptibility of human cells to Stx.

Hepatogastroenterology, 2003 Sep-Oct, 50(53), 1301 - 4
Peripheral blood mononuclear cells are possible extrahepatic replication sites for hepatitis C virus; Nishiguchi S et al.; BACKGROUND/AIMS: Hepatitis C virus is a major causative agent of chronic liver disease and hepatocellular carcinoma and is considered to be a hepatotropic virus . It remains controversial whether hepatitis C virus exists in peripheral blood mononuclear cells and replicates there . In order to resolve this issue, we performed nested RT-PCR (reverse transcription polymerase chain reaction) and RT-PCR in situ hybridization in peripheral blood mononuclear cells of patients with chronic hepatitis C . METHODOLOGY: We collected peripheral blood mononuclear cells from patients with chronic hepatitis C, extracted total RNA from the samples, and performed nested RT-PCR to detect hepatitis C virus RNA in the peripheral blood mononuclear cells lysates . We also fixed peripheral blood mononuclear cells of the patients in 4% paraformaldehyde and performed RT-PCR in situ hybridization with a digoxigenin-labeled RNA probe to detect hepatitis C virus RNA in the cells . RESULTS: Using these methods, we detected both positive- and negative-stranded hepatitis C virus RNA in peripheral blood mononuclear cells of hepatitis C patients . To determine in which cell population of peripheral blood mononuclear cells hepatitis C virus is present, we performed PCR in situ hybridization after incubation with fluorescent latex microbeads which could be phagocytozed by monocytes . We obtained positive signals of the replicative hepatitis C virus genome not only in lymphocytes but also in monocytes . CONCLUSIONS: RT-PCR in situ hybridization with a nonradioactive probe was found to be useful for in situ detection of hepatitis C virus RNA . Our findings suggest that peripheral blood mononuclear cells may be extrahepatic replication sites for hepatitis C virus.

Cell Motil Cytoskeleton, 2003 Nov, 56(3), 173 - 92
Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: neither microtubules nor actin filaments remain in the irradiated region; Forer A et al.; We studied chromosome movement after kinetochore microtubules were severed . Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region . After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving . We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy . For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation . Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions . Similarly, actin filaments normally present in kinetochore fibres were severed by the ultraviolet microbeam irradiations: the irradiated regions contained no actin filaments and only local spots of non-filamentous actin . There was no difference in irradiated regions when the associated chromosomes continued to move versus when they stopped moving . Thus, one cannot explain motion with severed kinetochore microtubules in terms of either microtubules or actin-filaments bridging the irradiated region . The data seem to negate current models for anaphase chromosome movement and support a model in which poleward chromosome movement results from forces generated within the spindle matrix that propel kinetochore fibres or kinetochore stubs poleward .

Appl Microbiol Biotechnol, 2004 Jan, 63(4), 351 - 61 Epub 2003 Oct 18.
Environmental and cultural stimulants in the production of carotenoids from microorganisms; Bhosale P; Commercial production of carotenoids from microorganisms competes mainly with synthetic manufacture by chemical procedures . Efficient stimulation of carotenoid biosynthesis is expected to promote accumulation of carotenoid by microbes . This review describes the variety of environmental and cultural stimulants studied during the last few decades which enhance volumetric production and cellular accumulation of commercially important carotenoids from microalgae, fungi and bacteria . Stimulation of carotenoid production by white-light illumination and temperature fluctuation is discussed along with supplementation of metal ions, salts, organic solvents, preformed precursors and several other chemicals in the culture broth . Reports on the improvements in yield are reviewed and assessed from a biotechnology point of view.

Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12899 - 904 Epub 2003 Oct 17.
Immunodominance among herpes simplex virus-specific CD8 T cells expressing a tissue-specific homing receptor; Koelle DM et al.; The study of immunodominance within microbe-specific CD8 T cell responses has been challenging . We used a previously undescribed approach to create unbiased panels of CD8 cytotoxic T lymphocyte clones specific for herpes simplex virus type 2, a pathogen with a complex genome encoding at least 85 polypeptides . Circulating herpes simplex virus type 2-specific cells were enriched and cloned after sorting for expression of the skin homing-associated receptor, cutaneous lymphocyte-associated antigen, bypassing restimulation with antigen . The specificity of the resultant cytotoxic clones was determined . Clonal frequencies were compared with each other and with the total number of cytotoxic clones . For each subject within the homing receptor-positive compartment, the CD8 cytotoxic response was dominated by T cells specific for only a few peptides . Previously undescribed antigens and epitopes in viral tegument, capsid, or scaffold proteins were immunodominant in some subjects . Clone enumeration analyses were confirmed in some subjects with dominance studies by using herpes simplex mutants, vaccinia recombinants, and/or enzyme-linked immune spots . We conclude that among circulating cells expressing a homing-associated receptor, during chronic herpes type 2 infection, the CD8 T cell response becomes quite focused despite the presence of many potential antigenic peptides.

Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12830 - 5 Epub 2003 Oct 17.
Proteorhodopsin genes are distributed among divergent marine bacterial taxa; de la Torre JR et al.; Proteorhodopsin (PR) is a retinal-binding bacterial integral membrane protein that functions as a light-driven proton pump . The gene encoding this photoprotein was originally discovered on a large genome fragment derived from an uncultured marine gamma-proteobacterium of the SAR86 group . Subsequently, many variants of the PR gene have been detected in marine plankton, via PCR-based gene surveys . It has not been clear, however, whether these different PR genes are widely distributed among different bacterial groups, or whether they have a restricted taxonomic distribution . We report here comparative analyses of PR-bearing genomic fragments recovered directly from planktonic bacteria inhabiting the California coast, the central Pacific Ocean, and waters offshore the Antarctica Peninsula . Sequence analysis of an Antarctic genome fragment harboring PR (ANT32C12) revealed moderate conservation in gene order and identity, compared with a previously reported PR-containing genome fragment from a Monterey Bay gamma-proteobacterium (EBAC31A08) . Outside the limited region of synteny shared between these clones, however, no significant DNA or protein identity was evident . Analysis of a third PR-containing genome fragment (HOT2C01) from the North Pacific subtropical gyre showed even more divergence from the gamma-proteobacterial PR-flanking region . Subsequent phylogenetic and comparative genomic analyses revealed that the Central North Pacific PR-containing genome fragment (HOT2C01) originated from a planktonic alpha-proteobacterium . These data indicate that PR genes are distributed among a variety of divergent marine bacterial taxa, including both alpha- and gamma-proteobacteria . Our analyses also demonstrate the utility of cultivation-independent comparative genomic approaches for assessing gene content and distribution in naturally occurring microbes.

J Histochem Cytochem, 2003 Nov, 51(11), 1533 - 44
Vimentin-positive cells in the epithelium of rabbit ileal villi represent cup cells but not M-cells; Ramirez C et al.; Membranous (M)-cells are specialized epithelial cells of the Peyer's patch domes that transport antigens from the intestinal lumen to the lymphoid tissue . Vimentin is a reliable marker for M-cells in rabbits . Using immunohistochemistry (IHC), a subpopulation of epithelial cells has recently been identified in ordinary rabbit ileal villi, which are vimentin-positive and share morphological characteristics with the M-cells of the domes . To test the hypothesis that these cells represent M-cells outside the organized lymphoid tissue, lectin labeling and tracer uptake experiments were performed . Lectins specific for N-acetyl-glucosamine oligomers selectively bound to the vimentin-positive villous cells but not to M-cells in the domes . Microbeads instilled into the ileal lumen were taken up by M-cells within 45 min but not by the vimentin-positive cells in the villi . Lectin-gold labeling on ultrathin sections revealed that the lectin binding sites were located in the brush border and in vesicles in the apical cytoplasm . The vimentin/lectin-positive cells shared ultrastructural characteristics with the so-called "cup cells." We conclude (a) that the vimentin-positive cells in ordinary villi represent cup cells but not M-cells, (b) that they are readily detectable by (GlucNAc)(N)-specific lectins, and (c) that they do not transcytose experimental tracers . Although the specific function of cup cells is still obscure, they most probably represent a cell type distinct from M-cells of the domes with respect to both function and expression of the two new markers.

Radiat Res, 2003 Nov, 160(5), 505 - 11
Low-dose studies of bystander cell killing with targeted soft X rays; Schettino G et al.; The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam . The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons . Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target . Circular diffraction gratings (i.e . zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position . Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically . The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range . The response when all cells were irradiated was compared to that obtained when only a single cell was exposed . The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses) . When only a single cell was targeted within the population, 10% cell killing was measured . In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy) . In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation . Damaged cells were distributed uniformly over the area of the dish scanned (approximately 25 mm2) . However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.

Radiat Res, 2003 Nov, 160(5), 512 - 6
Interaction between radiation-induced adaptive response and bystander mutagenesis in mammalian cells; Zhou H et al.; Two conflicting phenomena, the bystander effect and the adaptive response, are important in determining biological responses at low doses of radiation and have the potential to have an impact on the shape of the dose-response relationship . Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we reported previously that nonirradiated cells acquired mutagenesis through direct contact with cells whose nuclei had previously been traversed with either a single or 20 alpha particles each . Here we show that pretreatment of cells with a low dose of X rays 4 h before alpha-particle irradiation significantly decreased this bystander mutagenic response . Furthermore, bystander cells showed an increase in sensitivity after a subsequent challenging dose of X rays . Results from the present study address some of the pressing issues regarding both the actual target size and the radiation dose response and can improve on our current understanding of radiation risk assessment.

J Allergy Clin Immunol, 2003 Oct, 112(4), 686 - 94
Innate immune activation as a broad-spectrum biodefense strategy: prospects and research challenges; Hackett CJ; Biodefense strategies require protection against a broad and largely unforeseen spectrum of pathogens--the forte of innate immune system defenses--that have evolved over millennia to function within moments of encountering either ancient or newly emerging pathogens . Although constitutive, the innate immune system is activated by the presence of microbes or their products, providing a rationale for a potential biodefense strategy . Both prophylactic and postexposure strategies involving innate immune stimulation have been shown to be plausible to prevent or ameliorate infections in animal models . Innate immune-activating compounds based on conserved microbial components recognized by toll-like molecules and other receptors could be synthesized and delivered like drugs by using an entirely different strategy from conventional vaccination . However, important theoretic and practical questions emerge about developing and deploying innate immune protective strategies for biodefense . This rostrum discusses prospects and problems in the overall approach itself . Important topics include microbe-specific issues about innate immune system effectiveness against highly virulent pathogens and general questions, such as whether innate immune responses will be safe and effective if used in a diverse human population of different age groups and with different genetic makeups.

Biomaterials, 2003 Dec, 24(27), 4941 - 8
NMR properties of alginate microbeads; Simpson NE et al.; Alginates are a family of unbranched polysaccharides with properties that vary widely depending on their composition . In the presence of multivalent cations (frequently Ca2+), alginates form a gel . Consequently, alginates have been used to encapsulate a variety of biological materials, including cells . In this study, we present NMR relaxation and diffusion data from alginate microbeads with similar size and properties to those used in the development of a bioartificial pancreas . Our data demonstrate that the transverse relaxation time (T2) of water within the gel depends on the guluronic acid content of the alginate, whereas the longitudinal relaxation time (T1) and the apparent diffusion coefficient of water do not . Our data further suggest that the diffusion of Ca2+ ions is hindered by the presence of a poly-L-lysine layer, a layer commonly added to provide mechanical support to the beads and immunoprotection to the encapsulated cells in the event of implantation . The impact of these data on our understanding of the role of alginate gels in the development of a bioartificial pancreas is discussed.

Mol Plant Microbe Interact, 2003 Oct, 16(10), 926 - 35
A mating-induced protein of Phytophthora infestans is a member of a family of elicitors with divergent structures and stage-specific patterns of expression; Fabritius AL et al.; Five members of an elicitor-like gene family from Phytophthora infestans were examined . The family was identified through the analysis of M81, a mating-induced gene . The predicted M81 product resembled a 42-kDa P . sojae glycoprotein known to elicit defense reactions in plants, including a host of P . infestans, potato . M81 was the most structurally and functionally divergent of the P . infestans genes compared with the P . sojae sequence . M81 lacked elicitor activity, had the lowest protein identity (47%), displayed mating-specific transcription, and had a novel C-terminal domain . The latter contained a 30-residue proline- and threonine-rich motif, which, remarkably, was tandemly repeated 24 to 36 times in different alleles . M81C, M81D, and M81E better resembled the P . sojae protein based on amino acid identity (63 to 75%) and conserved elicitor activity . M81C and M81D mRNA accumulated only during zoosporogenesis, while M81E expression was restricted to hyphae . M81B, an apparent pseudogene, was physically linked to M81 . The protein products of each gene were predicted to be extracellular transglutaminases ranging in size from 436 to 1,607 amino acids . Genes with an elicitor, proline- and threonine-rich repeat, and both elicitor and repeat domains were widely distributed throughout Phytophthora infestans . These findings help explain the natural functions of elicitors in pathogen biology and plant-microbe interactions.

Oncogene, 2003 Oct 13, 22(45), 7043 - 9
Bystander responses induced by low LET radiation; Prise KM et al.; Radiation-induced bystander responses are observed when cells respond to their neighbours being irradiated . Considerable evidence is now available regarding the importance of these responses in cell and tissue models . Most studies have utilized two approaches where either a media-transferable factor has been assessed or cells have been exposed to low fluences of charged particles, where only a few percent are exposed . The development of microbeams has allowed nontargeted responses such as bystander effects to be more carefully analysed . As well as charged particle microbeams, X-ray microprobes have been developed, and several groups are also developing electron microbeams . Using the Gray Cancer Institute soft X-ray microprobe, it has been possible to follow the response of individual cells to targeted low doses of carbon-characteristic soft X-rays . Studies in human fibroblasts have shown evidence of a significant radiation quality-dependent bystander effect, measured as chromosomal damage in the form of micronuclei which is radiation quality dependent . Other studies show that even under conditions when only a single cell is targeted with soft X-rays, significant bystander-mediated cell killing is observed . The observation of bystander responses with low LET radiation suggests that these may be important in understanding radiation risk from background levels of radiation, where cells observe only single electron track traversals . Also, the indirect evidence for these responses in vivo indicates that they may have a role to play in current radiotherapy approaches and future novel strategies involving modulating nontargeted responses.

Oncogene, 2003 Oct 13, 22(45), 7034 - 42
Genomic instability and bystander effects induced by high-LET radiation; Hall EJ et al.; An understanding of the radiobiological effects of high-linear energy transfer (LET) radiation is essential for radiation protection and human risk assessment . Ever since the discovery of X-rays was made by Rontgen more than a century ago, it has always been accepted that the deleterious effects of ionizing radiation, such as mutation and carcinogenesis, are due mainly to direct damage to DNA . With the availability of a precision single-particle microbeam, it is possible to demonstrate, unequivocally, the presence of a bystander effect with many biological end points . These studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha-particles, and that cell-cell communication processes play a critical role in mediating the bystander phenomenon . Following exposure to high-LET radiation, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells have been shown to undergo malignant transformation through a series of successive steps, before becoming tumorigenic in nude mice . There is a progressive increase in genomic instability, determined either by gene amplification or allelic imbalance, with the highest incidence observed among established tumor cell lines, relative to transformed, nontumorigenic and control cell lines.

Clin Microbiol Rev, 2003 Oct, 16(4), 673 - 87
Bacterial wall as target for attack: past, present, and future research; Koch AL; When Bacteria, Archaea, and Eucarya separated from each other, a great deal of evolution had taken place . Only then did extensive diversity arise . The bacteria split off with the new property that they had a sacculus that protected them from their own turgor pressure . The saccular wall of murein (or peptidoglycan) was an effective solution to the osmotic pressure problem, but it then was a target for other life-forms, which created lysoymes and beta-lactams . The beta-lactams, with their four-member strained rings, are effective agents in nature and became the first antibiotic in human medicine . But that is by no means the end of the story . Over evolutionary time, bacteria challenged by beta-lactams evolved countermeasures such as beta-lactamases, and the producing organisms evolved variant beta-lactams . The biology of both classes became evident as the pharmaceutical industry isolated, modified, and produced new chemotherapeutic agents and as the properties of beta-lactams and beta-lactamases were examined by molecular techniques . This review attempts to fit the wall biology of current microbes and their clinical context into the way organisms developed on this planet as well as the changes arising since the work done by Fleming . It also outlines the scientific advances in our understanding of this broad area of biology.

Virology, 2003 Sep 30, 314(2), 671 - 9
Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells; Rojas OL et al.; Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals . In children with acute RV infection, these T cells were barely or not detectable . In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection . Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children . Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults . Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin . In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7 . These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.

Pharmacol Ther, 2003 Oct, 100(1), 75 - 87
Why are dendritic cells important in allergic diseases of the respiratory tract?
Upham JW, Stumbles PA.
Increasing evidence points to the role of antigen-presenting dendritic cells (DC) in regulating adaptive immune responses . DC are especially sensitive to signals derived from microbes, allergens, and the airway tissue microenvironment, can polarize naive T-cells into either Th1 or Th2 effector cells, and are increasingly recognized as having a central role in the establishment of T-cell memory and tolerance to inhaled antigens . DC form a closely meshed network within the respiratory mucosa and are rapidly recruited from the circulation in response to a variety of proinflammatory stimuli . Studies using animal models have highlighted the role of DC in both initiation and maintenance of allergic airway inflammation . Increased numbers of airway mucosal DC are found in both allergic rhinitis and asthma, and an increasing number of investigators have highlighted important functional differences between DC from atopic and normal individuals . This article reviews recent information on the involvement of DC in the pathogenesis of allergic airway disease and the means by which DC could be exploited as targets for therapy in asthma and allergic rhinitis.

Biotechnol Adv, 1991, 9(2), 157 - 71
Field testing of genetically engineered microorganisms; Drahos DJ; The first approved field releases of microorganisms genetically altered in the laboratory have been initiated in the past several years . While most introductions have been carried out in the United States, several tests have also occurred in the United Kingdom and Australia . Although such releases remain controversial in some areas, these pioneering studies have provided significant insight into the environmental behavior and relative safety of applying these microbes in a well-planned and carefully monitored program.

Biotechnol Adv, 1983, 1(1), 73 - 80
Biotechnology applied to mining of metals; Torma AE; The present review describes the advances achieved during the last two years in the application of biotechnological principles in the extraction of metals from ores and minerals . Despite the fact that this branch of science is very young and many details are yet to be understood, the microbes are applied at commercial levels especially for the extraction of copper and uranium from low-grade ores . The technique is far from being developed to its full potential and it is generally recognized to be a technology of the future . The studies involved are complex and multidisciplinary in nature.

Biotechnol Adv, 1983, 1(2), 289 - 300
Protoplast fusion as a means of producing new industrial yeast strains; Scheinbach S; The ability of the yeast Saccharomyces cerevisiae to produce ethanol and carbon dioxide from carbohydrates has been exploited by man for thousands of years . During its brief existence protoplast fusion has already become an invaluable tool for investigating the molecular genetics of yeast, as well as an important part of the arsenal of genetic manipulations available to develop new strains . In the case of industrial strains, a mating reaction is usually lacking . Protoplast fusion overcomes this barrier and allows for the genetic analysis of commercially valuable traits . A major block toward broader applicability of fusion is that hybrids becomes more unstable as the genetic backgrounds of the parents diverge . As greater progress in overcoming this problem is made, fusion, by itself and in conjunction with classical hybridization, will become increasingly important in the development of new strains . The incorporation of cytoplasmic elements into yeast protoplasts has the potential to vastly expand the array of biochemical reactions performed by yeasts, thereby increasing the importance of this microbe to mankind.

Biotechnol Adv, 1995, 13(2), 235 - 46
Prospects for the genetic manipulation of dairy cattle: opportunities beyond BST; Jones DD et al.; The dairy industry, with regulatory approvals of recombinant chymosin and bovine somatotropin (BST), has been at the forefront of food and agricultural biotechnology . The commercial fate of these products is one of several factors that may affect the success of future genetic manipulations in dairy cattle and dairy products . Other factors include technical and reproductive constraints in cattle and the cost of producing transgenic cattle . Early applications of genetic manipulation in cattle, for reasons of cost recoupment, may favor production of heterologous proteins in milk for pharmaceutical and medical use . Such applications could benefit genetic modification of milk and milk proteins for food use by providing advance knowledge and experience in mammalian protein expression . Other research opportunity areas that could affect prospects for genetic manipulation of dairy cattle include genome mapping, metabolic pathways, growth and development, and cattle/microbe interactions.

Adv Exp Med Biol, 2001, 491, 431 - 43
Pathogen-host protein-carbohydrate interactions as the basis of important infections; Karlsson KA; Microbe-host protein-carbohydrate interactions are most likely the essential first step to produce an infection, although this has been strictly proven only in a few cases . Improved glycotechnology will help identification of new carbohydrate receptors and this knowledge may be used to identify microbial carbohydrate-binding proteins by affinity proteomics approaches . In some cases such conserved proteins may prove to be successful vaccine components, in other cases, like influenza, saccharide analogues may be the only rational alternative . The prognosis may be, based on these improvements, that infection medicine will make considerable progress in the near future.

Water Sci Technol, 2003, 48(4), 113 - 9
Population dynamics of rumen microbes using modern techniques in rumen enhanced solid incubation; Raizada N et al.; The microbial ecology of the rumen is very complex . Different species of bacteria, protozoa, and fungi are involved in digestion of plant material in ruminants . In spite of complicated interrelationships among the various groups of microorganisms in the rumen ecosystem, Bacteria and Archaea are believed to play a major role because of their numerical predominance and metabolic diversity . In this work we are presenting the results for microbial population dynamics of rumen microbes during two-stage anaerobic digestion of grass . The reactors were inoculated with fresh rumen content . Fluorescent in situ hybridization, confocal laser scanning microscopy and epifluorescence microscopy were employed for microbial investigation . It was observed that Bacteria dominated in the hydrolytic reactor (1st stage) whereas Archaea were predominant in the methanogenic reactor (2nd stage) . The stability of the methanogenic reactor was result of the dominance of Methanosaeta species (mainly the filamentous type).

Int J Food Microbiol, 2003 Nov 1, 87(3), 207 - 16
Inactivation of microbes using ultrasound: a review; Piyasena P et al.; Alternative methods for pasteurization and sterilization are gaining importance, due to increased consumer demand for new methods of food processing that have a reduced impact on nutritional content and overall food quality . Ultrasound processing or sonication is one of the alternative technologies that has shown promise in the food industry . Sonication alone is not very effective in killing bacteria in food; however, the use of ultrasound coupled with pressure and/or heat is promising . Thermosonic (heat plus sonication), manosonic (pressure plus sonication), and manothermosonic (heat and pressure plus sonication) treatments are likely the best methods to inactivate microbes, as they are more energy-efficient and effective in killing microorganisms . Ultrasonic processing is still in its infancy and requires a great deal of future research in order to develop the technology on an industrial scale, and to more fully elucidate the effect of ultrasound on the properties of foods.

Nat Prod Res, 2003 Oct, 17(5), 369 - 73
A comparative study of essential oils of Cymbopogon citratus and some members of the genus Citrus; Saleem M et al.; Steam distilled oils of some species of the genus Citrus and Cymbopogon citratus were analyzed by GC-MS . It is observed that citral b was the most common constituent of the oils, which could be a good inhibitor of beta-glucuronidase as all the tested essential oils showed significant inhibition of beta-glucuronidase . IC50 values of a mixture of citral a and b also proved the hypothesis . The same oils also exhibited positive response against tested microbes.

Immunol Allergy Clin North Am, 2003 Aug, 23(3), 337 - 55
Sampling devices; Muilenberg ML; A number of commonly used samplers are presented in this article . Many samplers have not been discussed because they are used for specific purposes or are considered research tools . Air sampling for microbes may seem like a simple proposal, yet to develop and implement a well-thought out plan that answers questions or hypotheses with a high level of reliability is often a difficult and expensive undertaking . Sampler selection is only one step in this process . The information given in this article, along with the other resources listed, should aid in setting up a useful bioaerosol sampling plan.

Vasa, 2003 Aug, 32(3), 155 - 8
Vacuum-sealed mesh graft transplantation in chronic cutaneous ulcers of the lower leg; Halter G et al.; BACKGROUND: The covering of defects caused by chronic ulcers on limbs affected by peripheral arterial disease or chronic venous insufficiency is often difficult due to extensive secretion and edema, while chronic bacterial contamination of the wound bed further compromises the conditions for successful healing . PATIENTS AND METHODS: Vacuum-sealed dressing (VSD) offers the option of a closed dressing system for moist wound care that assures firm contact with the wound surface and protection against contamination with nosocomial microbes and decontamination of existing bacteria by means of constant drainage of secretion independent of gravity . VSD is particularly useful in difficult wounds featuring extensive secretion and unfavorable localization and offers many advantages over conventional dressing techniques in terms of improved healing of skin transplants . A total of 35 patients with chronic leg ulcers were treated with vacuum-sealed mesh graft transplantation . RESULTS: Complete healing of the mesh graft transplant was observed in 20 patients (57%) . Twelve patients (34%) experienced partial healing (75-90%) of the transplant, while three patients exhibited less than 75% healing of the graft and therefore required a second mesh graft transplantation . CONCLUSIONS: VSD is a simple, quick and inexpensive technique that promotes excellent healing of skin transplants . It is particularly useful in difficult wounds with extensive secretion and/or contamination or infection.

J Mol Recognit, 2003 Sep-Oct, 16(5), 318 - 23
Targeting with scFv: immune modulation by complement receptor specific constructs; Molnar E et al.; The fate of a microbe in the host is determined by various molecules of the innate immune system, which recognize the microbe and enhance its interaction with antigen presenting cells . This 'natural targeting' phenomenon, however, does not function when antigens with limited immunogenicity enter the host . Peptide vaccines, for instance, require adjuvants to induce immune responses . As a surrogate for the natural targeting mechanisms, antibodies against selected receptors of antigen presenting cells, conjugated with the peptides, could be used as targeting devices . Here we review various antibody-mediated antigen-targeting strategies, paying special attention to complement receptor-mediated targeting . We also describe and summarize our method of single-chain antibody-mediated targeting of viral epitopes to complement receptor type two and discuss the perspectives of single-chain antibody-mediated antigen targeting .

Novartis Found Symp, 2003, 251, 129 - 43; discussion 144-8, 281-97
Microbiology and immunology of autism spectrum disorders; Lipkin WI et al.; Both generic and environmental factors are likely to contribute to the pathogenesis of neurodevelopmental disorders . Even in heritable disorders of high penetrance, variability in timing of onset or severity of disease indicate a role for modifying principles . Investigation in animal models of the consequences of interactions between host response genes and microbes, toxins, and other environmental agents in a temporal context may elucidate the pathophysiology of a wide spectrum of chronic diseases . Here we review the evidence that infectious and immune factors may contribute to the pathogenesis of neurodevelopmental disorders, describe an animal model of neurodevelopmental disorders based upon viral infection, identify processes by which neural circuitry may be compromised, and outline plans for translational research in animal models and prospective human birth cohorts.

Clin Exp Allergy, 2003 Oct, 33(10), 1330 - 5
CpG DNA and immunotherapy of allergic airway diseases; Jain VV et al.; Atopic asthma is a highly prevalent and serious health problem for which no therapy currently offers the hope of a cure . Preindustrialized and rural populations appear relatively protected from the asthma epidemic; the hygiene hypothesis ascribes this protection to the effects of microbes and microbial products . An important immunostimulant component of microbes is DNA; bacterial DNA contains sequence motifs centred on the CpG dinucleotide, which are suppressed in mammalian DNA . Oligonucleotides containing these motifs (CpG ODN), like bacterial DNA, promote Th1 and regulatory-type immune responses . Using CpG ODN, we and others have demonstrated in murine studies that CpG ODN are effective in preventing the development of atopic airways disease . Moreover, when administered in conjunction with experimental allergen, they promote the reversal of established eosinophilic inflammation . These data suggest that CpG ODN may be a novel therapeutic tool for the treatment of atopic asthma.

J Refract Surg, 2003 Sep-Oct, 19(5), S597 - 601
From scattering to wavefronts--what's in between?
Mrochen M, Semchishen V.
PURPOSE: Some optical errors are too localized and random to be detected by commercial wavefront devices and Zernike polynomial expression . We looked beyond aberrations defined by Zernike expression to discuss implications of fine irregularities associated with highly aberrated corneal surfaces and complex surface roughness that can lead to light scattering . METHODS: Most fine irregularities are related to postoperative surface roughness, complexities of corneal ablation, and the laser in situ keratomileusis (LASIK) flap . These can be characterized mathematically by a random function that includes local surface tilts, the correlation radius of irregularities (Ic), surface roughness, and other terms . The Kirchoff method of scatter analysis characterizes fine surface irregularities by replacing each point on the surface with a tangential plane, allowing it to be governed by Snellen and Fresnel laws . RESULTS: The joint action of the continuum of microbeams defines a complex point spread function that can be expressed by the Strehl ratio . Small, highly irregular steep central islands and flap striae may not be adequately detected by Zernike expression and may have a surface irregularity diameter of 0.1 to 2.0 mm and height of 10 to 20 microm that results in a reduced Strehl ratio below 0.8 . Laser ablation inhomogeneities may have dimensions of 1 to 10 microm, resulting in a root mean square tilt value approaching 1.0 and a Strehl ratio below 0.5 . CONCLUSION: Corneal surface irregularities after laser vision correction may induce significant optical aberrations and distortions apart from classical wavefront or scattering errors . As these may not be detected by commercial wavefront devices, and yet contribute to the degradation of optical performance, alternate techniques should be evaluated to detect and describe these surface irregularities.

Biotechnol Lett, 2003 Aug, 25(16), 1357 - 61
A microbial fuel cell with improved cathode reaction as a low biochemical oxygen demand sensor; Kang KH et al.; Mediator-less microbial fuel cells (MFC) enriched with oligotrophic microbes were optimized through enhancement of cathode reaction and lowering O2 diffusion into the anode compartment as a low BOD sensor . The optimization of the MFC has greatly improved the maximum current and coulomb yield . The oligotroph-type MFC could be used as a low BOD sensor with high operational stability, good repeatability and reproducibility.

Oecologia, 2003 Dec, 137(4), 603 - 9 Epub 2003 Sep 26.
Soil community composition and the regulation of grazed temperate grassland; Frank DA et al.; The effect of the community composition of soil microbes on ecosystem processes has received relatively little attention . Here we examined the variation in soil microbial composition in a Yellowstone National Park grassland and the effect of that variation on the growth, in a greenhouse, of the dominant grass in the community . Plants and their rhizospheric soil were collected from paired, Poa pratensis-dominated grassland plots located inside and outside a 40-year-old exclosure . P . pratensis aboveground, belowground, and whole plant growth were greater in pots with soil communities from grazed grassland compared to fenced grassland, indicating (1) soil microbial communities differed, and (2) this difference influenced the growth of the plant that dominated both grasslands . Treating pots with fungicide (benomyl) suppressed the soil community influence, indicating that different fungal communities caused the soil microbe effect . In addition, two lines of evidence are consistent with the hypothesis that arbuscular mycorrhizal fungal (AMF) species composition affected P . pratensis: (1) a divergence in AMF spore communities in the two field soils, and (2) little evidence of pathogenic fungi . These findings emphasize the need to examine the role that the composition of the soil microbial community plays in controlling terrestrial ecosystems.

Leukemia, 2003 Oct, 17(10), 1973 - 80
Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy; Guven H et al.; B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world . It is currently an incurable disease, making new treatment options such as immunotherapy desirable . Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option . Administration of cytotoxic cells such as natural killer (NK) and natural killer-like T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs . A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells . Here, we report the feasibility of expanding populations of the human killer cells, CD3-CD56+ NK and CD3+CD56+ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients . The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture . The 21-day cultures from both B-cell- and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype . Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly . In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment . Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates . Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B . From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.

Environ Microbiol, 2003 Oct, 5(10), 828 - 41
New primers for the class Actinobacteria: application to marine and terrestrial environments; Stach JE et al.; In this study, we redesigned and evaluated primers for the class Actinobacteria . In silico testing showed that the primers had a perfect match with 82% of genera in the class Actinobacteria, representing a 26-213% improvement over previously reported primers . Only 4% of genera that displayed mismatches did so in the terminal three bases of the 3' end, which is most critical for polymerase chain reaction success . The primers, designated S-C-Act-0235-a-S-20 and S-C-Act-0878-a-A-19, amplified an approximately 640 bp stretch of the 16S rRNA gene from all actinobacteria tested (except Rubrobacter radiotolerans) up to an annealing temperature of 72 degrees C . An Actinobacteria Amplification Resource was generated to provide a visual guide to aid the amplification of actinobacterial 16S rDNA . Application of the primers to DNA extracted from marine and terrestrial samples revealed the presence of actinobacteria that have not been described previously . The use of 16S rDNA similarity and DNA-DNA pairing correlations showed that almost every actinomycete clone represented either a new species or a novel genus . The results of this study reinforce the proposition that current culture-based techniques drastically underestimate the diversity of Actinobacteria in the environment and highlight the need to evaluate taxon-specific primers regularly in line with improvements in databases holding 16S rDNA sequences.

J AOAC Int, 2003 Jul-Aug, 86(4), 737 - 45
Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison; Ruberu SR et al.; A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting . The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter . Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument . Precision, accuracy, and sensitivity {limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue} of the modified protocol were equivalent to those of the original protocol . The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values . Comparison of STX reference standards obtained from the U.S . Food and Drug Administration and the National Research Council, Canada, showed no observable differences . This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

Invest Ophthalmol Vis Sci, 2003 Oct, 44(10), 4505 - 14
P2Y(2) receptor agonist INS37217 enhances functional recovery after detachment caused by subretinal injection in normal and rds mice; Nour M et al.; PURPOSE . To evaluate the effects of INS37217 on the recovery of retinal function after experimental retinal detachment induced by subretinal injection . METHODS . Subretinal injections of 1 micro L of fluorescent microbeads, saline, or INS37217 (1-200 micro M) were made by the transvitreal method in normal (C57BL/6) mice and in mice heterozygous for the retinal degeneration slow (rds) gene . Control, mock-injected animals underwent corneal puncture without injection . Histologic and ERG evaluations were made at 0 to 1 and 8 hours, and 1, 3, 7, 10, 14, and 60 days post injection (PI) . DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) . RESULTS . A single subretinal injection of saline solution containing fluorescent beads caused a histologically evident retinal detachment and distributed the microbeads to almost all the subretinal space . Spontaneous reattachment occurred within 24 hours after injection and was accompanied by evident retinal folding that appeared largely resolved by 6 days later . Relative to controls, injection of saline resulted in approximately 40% recovery of dark-adapted a-wave amplitude at 24 hours PI and gradually improved to approximately 90% of controls at 2 months PI . Subretinal injection of saline containing INS37217 (10 micro M) significantly increased rod and cone ERG of normal and rds(+/-) mice at 1 and 10 days PI, when compared with injection of saline alone . Additionally, INS37217 reduced the number of TUNEL-positive photoreceptors and the enhanced rate of reattachment . CONCLUSIONS . Enhancement of ERG recovery by INS37217 is likely due to reduced retinal folding and cell death associated with detachment . These results support the use of INS37217 to help restore function after therapies that involve subretinal administration of drugs in animal models of retinal diseases.

Bioessays, 2003 Oct, 25(10), 930 - 9
Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms; Mattick JS; The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein . As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals . However, the latter may not be the case in complex organisms . A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect . ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing . This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoietic development and phenotypic variation .

Naturwissenschaften, 2003 Sep, 90(9), 406 - 9 Epub 2003 Aug 12.
The effect of metapleural gland secretion on the growth of a mutualistic bacterium on the cuticle of leaf-cutting ants; Poulsen M et al.; In Acromyrmex octospinosus leaf-cutting ants the metapleural glands produce an array of antibiotic compounds that serve as a general defence against unwanted microbes on the cuticle . Leaf-cutting ants also grow mutualistic Pseudonocardiaceae bacteria on their cuticle that produce antibiotics controlling a microfungal parasite of their fungus gardens . Interaction between this bacterium and gland secretion therefore seems unavoidable . We document the typical development of bacterial growth on the cuticle of young major workers, show that growth starts a few days after eclosion, and that the maximal cover is reached after 2-3 weeks and gradually declines when workers mature . Experimental closure of the metapleural glands had no effect on the initial exponential growth phase of the bacterium, but significantly reduced the cover during the decline phase . The age-dependent abundance of the bacterium and its partial dependence on metapleural gland secretion support the hypothesis that the abundance of this mutualist is actively regulated.

Curr Opin Allergy Clin Immunol, 2003 Oct, 3(5), 325 - 9
Environmental exposure to endotoxin and other microbial products and the decreased risk of childhood atopy: evaluating developments since April 2002; Braun-Fahrlander C; PURPOSE OF REVIEW: The hygiene hypothesis proposes an association between the change in exposure to microbes and the increased incidence of atopic diseases in recent decades . Exposure to microbes and childhood infections and their association with atopy has thus attracted much scientific interest . This review focuses on new developments in the field of epidemiology . RECENT FINDINGS: Studies in adults confirm that exposure to orofaecal pathogens are associated with less asthma and allergies . In seropositive individuals, no increase in allergy prevalence over time was noted . Similarly, the generational increase in atopy and allergic rhinitis was not observed in individuals who were exposed to a farming environment in childhood . More than 20 studies have been published examining the effect of exposure to a farm environment in children and adults . Most consistently, the 'protective' farm effect was related to livestock farming and thus to microbial exposure . A dose-dependent inverse relationship between exposure to endotoxin in the mattress dust of children and the occurrence of atopic diseases was shown in rural environments in Europe . In addition, the blood cells of farmers' children were shown to express higher amounts of innate immunity receptors . Only a few farm studies have so far included an objective measure of the microbial environment . The examined exposure to endotoxin might partly be a surrogate measure of a much broader spectrum of immunomodulatory microbial compounds present in a rural environment . SUMMARY: The 'hygiene hypothesis' has gained much credibility, but the results should be balanced against the benefits of established hygiene standards.

Methods Mol Biol, 2003, 236, 79 - 84
Combined ESTs from plant-microbe interactions: using GC counting to determine the species of origin; Huitema E et al.; A diversity of microorganisms establishes intimate associations with plants . Whether pathogenic or symbiotic, such interactions are the result of complex recognition events between plants and microbes, leading to signaling cascades and regulation of countless genes involved in the interaction . A key step in unraveling the mysteries of plant-microbe interactions lies in defining the transcriptional changes that occur in both the host and the microbe during their association . The sum of the transcripts, from both host and microbe, which are produced during their association, has been defined as the interaction transcriptome . One approach to analyze interaction transcriptomes is to perform large-scale sequencing of cDNAs (expressed sequence tags or ESTs) obtained from infected plant tissue and representing a mixture of host and microbe sequences . In some cases, the two organisms have markedly different GC content, allowing most ESTs to be easily distinguished on this basis . In this chapter, we describe a GC counting method to determine the species of origin of ESTs obtained from interactions between plants and oomycetes or other high GC content microbes.

J Microbiol Methods, 2003 Oct, 55(1), 325 - 9
A microplate fluorimetric assay for measuring dehalogenase activity; Marchesi JR; A fluorimetric assay was developed to measure halide release from halogenated compounds being degraded by microbes . The method relies on the property of halides to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) by collision quenching . The assay shows a wide response to halide concentration (1-500 mM) and tolerates a wide pH range . Furthermore, it is simple to use, has the potential for automation and uses an inexpensive non-toxic reagent.

Curr Opin Immunol, 2003 Oct, 15(5), 560 - 5
Granulysin; Clayberger C et al.; Granulysin, a molecule expressed by human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of microbes and tumors . Progress in understanding the structure, function and clinical relevance of granulysin over the past year encompasses three main areas: first, the solution of its crystal structure, providing new insights into its potential mechanism of target cell damage; second, inhibition of its function with small interfering RNA, indicating its relevance in microbial immunity; and third, association of granulysin expression in natural killer cells with good outcomes in cancer, indicating its potential utility as a diagnostic and suggesting its relevance to human disease.

Biochem J, 2003 Dec 1, 376(Pt 2), 313 - 38
Structure and function of efflux pumps that confer resistance to drugs; Borges-Walmsley MI et al.; Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact . From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic . Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases . Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs . This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms . A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell . Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion . Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates . These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids . If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation . These are key questions that must be addressed . In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps.

Opt Lett, 2003 Sep 15, 28(18), 1654 - 6
Multiple imaging axis microscopy improves resolution for thick-sample applications; Swoger J et al.; The multiple imaging axis microscope (MIAM) is a wide-field optical microscope that observes a sample simultaneously from multiple directions without requiring the sample to be rotated or tilted . The prototype is capable of high-resolution imaging of the interior of a 300-microm-diameter sample consisting of fluorescent microbeads suspended in an agarose gel . Compared with a single-axis system, the MIAM can achieve a reduction of the axial point-spread function elongation by a factor of 5.8 and a 3.5-fold improvement in volume resolution by simple linear image combination techniques.

Clin Infect Dis, 2003 Oct 1, 37(7), 939 - 42 Epub 2003 Sep 05.
Infectious diseases in cinema: virus hunters and killer microbes; Pappas G et al.; The world of infectious diseases has been rarely presented in the cinema with accuracy . Apart from random biographies of scientists and retellings of stories about great epidemics from the past, most films focus on the dangers presented by outbreaks of unknown agents that originate from acts of bioterrorism, from laboratory accidents, or even from space . We review these films and underline the possible effect that they have on the public's perception of infection--a perception that, when misguided, could prove to be problematic in times of epidemics.

APMIS, 2003 Jul-Aug, 111(7-8), 789 - 96
Dendritic cells and fungi; Buentke E et al.; Fungi comprise a group of microorganisms that in the past 20 years has become increasingly important as a cause of human disease . Few fungi are professional but instead opportunistic pathogens, and some fungi can even act as allergens . Dendritic antigen-presenting cells function as a link between innate and adaptive immunity and are therefore important in recognition of pathogens . Effective defense requires the host to discriminate between different pathogens to induce an appropriate response . Signaling from different groups of microbes can be mediated via the Toll-like receptors (TLRs), leading to activation of conserved host defense signaling pathways that control the expression of a variety of immune response genes . Different dendritic cells (DCs) express different patterns of recognition molecules, which indicate that they are more or less efficient when responding to certain pathogens . DCs have an important role in the induction of cell-mediated immune responses to fungi, and the studies reviewed here show that fungi, or possibly fungi-derived factors, provide a powerful activation stimulus to DCs, resulting in DC maturation with upregulation of co-stimulatory molecules and production of cytokine patterns leading to different T cell responses . The possibility of using ex vivo-generated DCs as therapeutic tools for restoring anti-fungal immunity is a challenge for the future.

Ying Yong Sheng Tai Xue Bao, 2003 Jun, 14(6), 1023 - 5
{Degradation of fenpropathrin, phoxim and their mixture by soil microbes}; Zhu L et al.; The degradation rates of fenpropathrin, phoxim and their mixture in un-sterilized soil were much quicker than those in sterilized soil, which indicated that soil microorganisms played a significant role in the degradation process in soil . The half-life (T0.5) in un-sterilized soil was 56.2 d for fenpropathrin, 57.8 d for mixed fenpropathrin, 48.2 d for phoxim, and 41.7 d for mixed phoxim . The corresponding half-life (T0.5) in sterilized soil was 135.1 d, 147.3 d, 123.6 d, and 126.2 d, respectively . There were no significant differences for degradation rates between single use and mixed use of fenpropathrin and phoxim.

Ying Yong Sheng Tai Xue Bao, 2003 Jun, 14(6), 1000 - 2
{Advances in studies on transported flux and properties of riverine organic carbon}; Gao Q et al.; Some lately advances in the study of riverine organic carbon were summarized in this paper . The transported flux of organic carbon from terrestrial ecosystems to the oceans via rivers, which is one of the most sensitive land surface processes in global climate change, has been changed in quantity due to the anthropogenic disturbance to it . The properties of riverine organic carbon, even in the same drainage, changed notably with the changes of hydrological processes in the drainage . Riverine organic carbon may become aged since they have been metabolized by the riverine microbes in the processes of being transported to the seas from the land, i.e., the radiocarbon in the riverine organic carbon was partially degraded by the microbes.

Vet Immunol Immunopathol, 2003 Sep 15, 95(1-2), 21 - 31
Maturation of bovine dendritic cells by lipopeptides; Hope JC et al.; The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge . The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells . We report here that bovine DC are also induced to mature following lipopeptide stimulation . Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFalpha . Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNgamma secretion . In contrast, exposure of MPhi to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion . A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFalpha secretion . These different changes in bovine DC and MPhi may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MPhi having a much less marked effect on immune responses.

Mol Gen Mikrobiol Virusol, 2003, (3), 25 - 8
{The role of lipopolysaccharide in toxicity of Francisella genus bacteria}; Onoprienko NN et al.; It was demonstrated that the lipopolysaccharides (LPS) preparations, which were isolated from all representatives of Francisella Genus bacteria, i.e . F . tularensis, F . novicida, F . novicida-like and F . philomiragia by using the method of R.P . Darveau, R.E . Hancock (1983), were not toxic for white rats and white mice . A comparative study of toxicity of live F . tularensis bacteria (both wild and LPS-defective strains) made it possible to establish a direct correlation between the toxicity of microbes and LPS chemotype . It was found that only typical strains, which synthesize the wild-type S-LPS, caused the death of white rats and white mice in 24 hours after intraperitoneal contamination (10(9), 10(10) CFU/animal) . Live bacteria of F tularensis R-mutants were not able to induce a lethal infection of rats and retained only residual virulence for mice . Other representatives of Francissela genus possessed less pronounced pathogenic properties . Thus, the toxic effect was registered, in case of white rats, only for F . novicida but not for F . novicida-like or F . philomiragia . At the same time, the two last mentioned species displayed a certain degree of virulence at high challenge doses (10(9), 10(10) CFU/animal) in respect to white mice . F . philomiragia, which generated lipoolygosaccharide (LOS) with an unusual structure, was found to be least pathogenic (25-75% of dead mice) . The toxicity of bacteria, killed experimentally by different means (heating, UV-light, chloroform, acetone and formalin), was studied to define the role of bacterial proteins in the realisation of F . tularensis toxic potential in vivo . No lethal effect was exerted on experimental animals by killed microbes or purified LPS preparations . Finally, the study results show a priority role of the LPS molecule in the toxic effect of F . tularensis, which is possible in vivo only if structurally valuable molecules of live bacterial cells are available.

Anal Chem, 2003 Jul 1, 75(13), 3161 - 7
Measurement of enzyme kinetics using a continuous-flow microfluidic system; Seong GH et al.; This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system . The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed . Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation . The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent . The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin . In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment . The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules . This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.

J Int Med Res, 2003 Jul-Aug, 31(4), 299 - 306
Comparison of the microbicidal activities of superoxidized and ozonated water in the disinfection of endoscopes; Urata M et al.; The microbicidal activities of superoxidized water (electrolysed strong acid water {ESAW} or electrolysed weak acid water {EWAW}), ozonated water, 0.05% chlorhexidine and 2% glutaraldehyde were tested against seven strains of clinical micro-organism isolates . Following incubation of bacterial suspensions in ESAW and EWAW for 10 s, the number of micro-organisms was reduced below the detection limit . The microbicidal activities of ESAW and EWAW were similar to that of glutaraldehyde, and superior to ozonated water and 0.05% chlorhexidine . The microbicidal activities of ESAW, EWAW and ozonated water were markedly diminished in the presence of albumin . Microbial contamination of upper gastrointestinal endoscopes was detected after 90 endoscopic procedures, but treatment of the endoscope with ESAW, EWAW or ozonated water eradicated the microbes . These results indicate that ESAW and EWAW are effective disinfectants after mechanical cleaning of upper gastrointestinal endoscopes, and can, therefore, be used in the endoscopy unit.

Med Sci Monit, 2003 Sep, 9(9), CR400 - 4
Characterization of Helicobacter pylori strainsisolated before and after therapy; Andrzejewska E et al.; BACKGROUND: Genotypic analysis has been performed in Helicobacter pylori strains isolated before and after antibiotic therapy from patients for whom no eradication of the microbes could be achieved . MATERIAL/METHODS: The Helicobacter pylori strains were isolated from 10 patients with duodenal ulcer, who were treated by the triple therapy (Controloc with amoxicillin and metronidazole) . Biopsies of gastric mucosa were inoculated on the Columbia agar with antibiotics and on the medium without antibiotics . The cultures were kept in microaerophilic conditions at 37 degrees C for 4 to 10 days . Genotype analysis was performed using PCR-based RAPD fingerprinting . The sensitivity of the strains to metronidazole, clarithromycin, amoxicillin and tetracycline was tested using RESULTS: In five patients, identical strains of the bacteria were noted both before and after the therapy while distinct patterns of pre- and post-treatment isolates were found in three patients . One patient demonstrated identical strains before and after the therapy but upon later control testing was found to be infected with a genotypically distinct strain . In 2 patients (a married couple) the infection was caused by genotypically distinct strains . A high proportion (46%) of metronidazole-resistant strains was detected . CONCLUSIONS: Therapeutic failures in treatment of Helicobacter pylori infections may either be the result of ineffective eradication of the strain from the stomach or of a re-infection . When applying treatment with antibiotics, high frequency of metronidazole resistance should be taken into

Mutagenesis, 2003 Sep, 18(5), 411 - 6
Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells; Tartier L et al.; Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity . In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells . We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair . We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX . Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells . This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms

J Leukoc Biol, 2003 Oct, 74(4), 479 - 85 Epub 2003 Jul 01.
How we detect microbes and respond to them: the Toll-like receptors and their transducers; Beutler B et al.; Macrophages and dendritic cells are in the front line of host defense . When they sense host invasion, they produce cytokines that alert other innate immune cells and also abet the development of an adaptive immune response . Although lipolysaccharide (LPS), peptidoglycan, unmethylated DNA, and other microbial products were long known to be the primary targets of innate immune recognition, there was puzzlement as to how each molecule triggered a response . It is now known that the Toll-like receptors (TLRs) are the principal signaling molecules through which mammals sense infection . Each TLR recognizes a restricted subset of molecules produced by microbes, and in some circumstances, only a single type of molecule is sensed (e.g., only LPS is sensed by TLR4) . TLRs direct the activation of immune cells near to and far from the site of infection, mobilizing the comparatively vast immune resources of the host to confine and defeat an invasive organism before it has become widespread . The biochemical details of TLR signaling have been analyzed through forward and reverse genetic methods, and full elucidation of the molecular interactions that transpire within the first minutes following contact between host and pathogen will soon be at hand.

Guang Pu Xue Yu Guang Pu Fen Xi, 2001 Dec, 21(6), 769 - 71
{Study on the deteriorating course of fresh milk by laser-induced fluorescence spectra}; Liu J et al.; Along with the development of living standard, people's demand for food quality and food hygiene also rises . People demand food not only with rich nutrition, inexpensive price, but also with safety . So food hygiene test is paid common attention of society . Milk is a nourishing food and is loved by people . Sour milk goods from milk is also in great demand . But nourishing foods are good for growing many microbes . Fresh milk and sour milk are easy contaminated by microbes and go bad . Laser-induced fluorescence (LIF) technology is an important part of modern optics . It is broadly applied in biomedicine, diagnostics, test of food hygiene, environment protecting, owing to its high sensitivity, high speed, automation, untouched testing . In this paper, we attempted to LIF technology to test milk food quality . We used the third harmonics pulsed Nd:YAG laser (355 nm) as the exciting source, and a multi-track spectrometer as the detector and measured the intensities of apply LIF of fresh milk and sour milk during their deteriorating course . Test system and test method are introduced, fluorescence spectra of deteriorating course are also attached . The test result makes clear that there are close connection between deteriorating course and fluorescence spectra.

J Clin Microbiol, 2003 Sep, 41(9), 4328 - 35
Isolation of an Anaplasma sp . organism from white-tailed deer by tick cell culture; Munderloh UG et al.; We used tick cell culture to isolate a bacterium previously referred to as the "white-tailed deer (WTD) agent" from two captive fawns inoculated with blood from wild WTD (Odocoileus virginianus) . Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34 degrees C, and 8 days later, Anaplasma-like inclusions were demonstrated in Giemsa-stained culture samples . The microbes became established and could be continuously passaged in tick cells . The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing . Comparison with sequences available in GenBank indicated that the isolate was most closely related first to Anaplasma platys and second to Anaplasma phagocytophilum, supporting its placement in the genus Anaplasma . Transmission electron microscopy of this Anaplasma sp . organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria . Tick cells infected with the Anaplasma sp . organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.

Appl Environ Microbiol, 2003 Sep, 69(9), 5603 - 8
Isolation and identification of actinobacteria from surface-sterilized wheat roots; Coombs JT et al.; This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.) . Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria . Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria . Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp . Many of the Streptomyces spp . were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp . that had been isolated from potato scabs . In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S . setonii . None of these isolates, nor the S . caviscabies and S . setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic . These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.

Methods, 2003 Oct, 31(2), 150 - 9
Detection and enrichment of antigen-specific CD4+ and CD8+ T cells based on cytokine secretion; Campbell JD; The Cytokine Secretion Assay is an innovative method for analysing and enriching live cytokine-secreting cells . In this assay, a cytokine affinity matrix is built on the cell plasma membrane, which traps cytokines produced by the cell in response to specific stimuli . The specifically bound cytokine is then detected, and the cells optionally enriched, using fluorochrome-conjugated cytokine-specific antibodies and magnetic microbeads . This method allows extremely detailed phenotyping of live cells and the detection of cytokine responses at very low frequencies . Here, the latest cell staining and separation procedures are reviewed, with particular reference to the best application of the technology and troubleshooting in a variety of different situations.

Australas Phys Eng Sci Med, 2003 Jun, 26(2), 63 - 9
Comparison of Monte Carlo simulations of photon/electron dosimetry in microscale applications; Joneja OP et al.; It is important to establish reliable calculational tools to plan and analyse representative microdosimetry experiments in the context of microbeam radiation therapy development . In this paper, an attempt has been made to investigate the suitability of the MCNP4C Monte Carlo code to adequately model photon/electron transport over micron distances . The case of a single cylindrical microbeam of 25-micron diameter incident on a water phantom has been simulated in detail with both MCNP4C and the code PSI-GEANT, for different incident photon energies, to get absorbed dose distributions at various depths, with and without electron transport being considered . In addition, dose distributions calculated for a single microbeam with a photon spectrum representative of the European Synchrotron Radiation Facility (ESRF) have been compared . Finally, a large number of cylindrical microbeams (a total of 2601 beams, placed on a 200-micron square pitch, covering an area of 1 cm2) incident on a water phantom have been considered to study cumulative radial dose distributions at different depths . From these distributions, ratios of peak (within the microbeam) to valley (mid-point along the diagonal connecting two microbeams) dose values have been determined . The various comparisons with PSI-GEANT results have shown that MCNP4C, with its high flexibility in terms of its numerous source and geometry description options, variance reduction methods, detailed error analysis, statistical checks and different tally types, can be a valuable tool for the analysis of microbeam experiments.

Am J Kidney Dis, 2003 Sep, 42(3), 575 - 81
Hypothesis: dysregulation of immunologic balance resulting from hygiene and socioeconomic factors may influence the epidemiology and cause of glomerulonephritis worldwide; Johnson RJ et al.; Glomerular diseases show diverse epidemiological characteristics throughout the world, which has been suggested to be caused by differences in genetics of the underlying populations or environmental exposure to the putative antigens or agents that either trigger or induce the disease . Recently, an alteration in immune balance of the T helper 1 (T(H)1) and T helper 2 (T(H)2) subsets has been implicated as a mechanism to explain the relative increase in allergic diseases in industrialized nations . According to the Hygiene Hypothesis, overcrowding and poor hygiene early in life may protect from atopic diseases because exposure to microbes predisposes in favor of a T(H)1-dominant response . Conversely, dominance of the T(H)2 subset would be responsible for the increasing incidence of allergies . We present the hypothesis that this imbalance may help explain the predilection for membranoproliferative glomerulonephritis (GN) and mesangial proliferative GN to be associated with developing and/or poor nations, whereas immunoglobulin A nephropathy and minimal change disease are observed more commonly in industrialized nations . The implication of the Hygiene Hypothesis is that clinical expression of immune-mediated renal disease would depend on the prevailing T(H)1/T(H)2 balance, rather than the etiologic agent, and it may help explain the epidemiological pattern of glomerular diseases worldwide.

Klin Khir, 2003 Jun, (6), 38 - 40
{Local medical support during surgical treatment of purulent inflammation}; Zheliba MD et al.; Acute purulent affection of soft tissues is revealing in 8.8-14% patients in the general surgery hospitals . Hospital strains of microbes of an acute surgical infection are highly sensitive toward preparations of surface--active substances, of which antiseptics on the decametoxine background have the highest antimicrobic activity . In 934 patients after surgical treatment of purulent wound the application of decametoxine and preparations on its background had promoted the treatment duration considerable decrease comparing the effect of the other antiseptics.

J Exp Med, 2003 Sep 1, 198(5), 693 - 704
Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment; Schnappinger D et al.; Little is known about the biochemical environment in phagosomes harboring an infectious agent . To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation . The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment . Genes expressed differentially as a consequence of intraphagosomal residence included an interferon gamma- and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon . Induction of genes involved in the activation and beta-oxidation of fatty acids indicated that fatty acids furnish carbon and energy . Induction of sigmaE-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope . Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice . The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.

J Agric Food Chem, 2003 Sep 10, 51(19), 5803 - 14
Trypsin/alpha-amylase inhibitors inactivate the endogenous barley/malt serine endoproteinase SEP-1; Jones BL et al.; Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases . It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins . It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase . Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme . Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions . Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions . When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not . This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase . It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.

J Perinat Med, 2003, 31(4), 287 - 94
Inducing proliferation of human amnion epithelial and mesenchymal cells for prospective engineering of membrane repair; Ochsenbein-Kolble N et al.; OBJECTIVE: To prepare a tissue engineering approach to fetal membrane repair after premature rupture of the membranes (PROM) by characterizing the proliferation potential of human amnion epithelial and mesenchymal cells from preterm and term placenta in primary culture . METHODS: Amnion epithelial and mesenchymal cells from 15 preterm (23-36 week) and 27 term placentas collected at cesarean section were separated enzymatically, characterized immunohistochemically (anti-cytokeratin 18 and anti-E-cadherin, and anti-vimentin, respectively), and their ratio determined . Proliferation on tissue culture polystyrene (TCPS) or collagen in one medium and on TCPS in four different media after 14 days was measured photometrically and compared in preterm vs . term placenta . For statistical analysis the Mann-Whitney test was used . RESULTS: Preterm and term epithelial:mesenchymal cell ratios were 4.3:1 and 7.8:1 . Term epithelial cells proliferated similarly on TCPS or collagen . Mesenchymal cells proliferated only with fetal bovine serum (FBS) . Proliferation of term amnion cells in medium containing FBS, epithelial growth factor (EGF), insulin, transferrin and triidothyronine(T3) was significantly increased (p < 0.001) compared with the other three media, and percentage proliferation was slightly higher in preterm cells . CONCLUSION: Characterization of human amnion epithelial and mesenchymal cells identified the most potent proliferation-inducing medium yet . Studies of the wound-healing potential of these cells are needed, examining their behavior and proliferation on fibrin microbeads and other extracellular matrixes as the next step towards engineering membrane repair in PROM.

J Leukoc Biol, 2003 Sep, 74(3), 331 - 43
Expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function; Melchjorsen J et al.; Recruitment and activation of leukocytes are important for elimination of microbes, including viruses, from infected areas . Chemokines constitute a group of bioactive peptides that regulate leukocyte migration and also contribute to activation of these cells . Chemokines are essential mediators of inflammation and important for control of viral infections . The profile of chemokine expression contributes to shaping the immune response during viral infection, whereas viral subversion of the chemokine system allows the virus to evade antiviral activities of the host . In this review, we discuss the role of chemokines in host-defense against virus infections, and we also look deeper into the virus-cell interactions that trigger chemokine expression as well as the cellular signaling cascades involved.

Am J Public Health, 2003 Sep, 93(9), 1489 - 93
Indoor environments and health: moving into the 21st century; Samet JM et al.; The quality of our indoor environments affects well-being and productivity, and risks for diverse diseases are increased by indoor air pollutants, surface contamination with toxins and microbes, and contact among people at home, at work, in transportation, and in many other public and private places . We offer an overview of nearly a century of research directed at understanding indoor environments and health, consider current research needs, and set out policy matters that need to be addressed if we are to have the healthiest possible built environments . The policy context for built environments extends beyond health considerations to include energy use for air-conditioning, selection of materials for sustainability, and design for safety, security, and productivity.

Pathol Biol (Paris), 2003 Sep, 51(7), 418 - 27
{Microbeads, nanobeads and cytometry: applications to the analysis and purification of cells and biomolecules}; Lizard G et al.; Nano and microspheres are important tools in cytometry . They have been used in first to optimize fluorescent signals detected by flow cytometry and to evaluate phagocytosis . Some antigens were also detected by using nanospheres covalently coupled to antibodies . Specifically dedicated microspheres are now widely used for antigenic quantitation by flow cytometry, and magnetic nano and micropheres are very usefull for cellular and molecular purifications . To date, analytical methods based on the use of microspheres are developed to detect proteins, nucleic acids, and ions . To this end, antibodies, oligonucleotides, or chelating agents are bound to microspheres characterized by different fluorescences . The applications of these multiplexed microspheres assays allow to identify and quantify simultaneously some macromolecules and ions, but they also permit to analyze enzymatic activities and to perform polymorphism analyses . With microspheres used as reactive support, molecular analyses are therefore possible by flow cytometry . Nano and microspheres are also usefull tools for calibration in confocal microscopy as well as for micromanipulations of biomolecules and of living cells . Inovative methods based on the use of nano and microspheres are expected in the fields of biology, medicine, food industry, and environmental sciences.

Am J Surg, 2003 Sep, 186(3), 301 - 5
Bacterial translocation: what it is and what it is not; Steinberg SM; The reasons our critically ill patients die have been a matter of great interest for many decades, with the hope that if we can identify the mechanisms responsible for death, we might be able to intervene and improve outcome . Over the last 1 to 2 decades, the concept of bacterial translocation-the movement of gut origin microbes across the intact gastrointestinal tract into normally sterile tissues where the organisms may then directly cause infection or incite an inflammatory response that causes tissue injury, organ failure, and death-has grown to the point where it is virtually impossible to make rounds in any intensive care setting without a resident or student blaming some complication or another on bacterial translocation . We will attempt to review the clinically relevant information that supports and refutes the concept of bacterial translocation as a cause of our critically ill patients to exhibit the symptoms and signs of sepsis, develop organ failure, and ultimately die.

Phytochemistry, 2003 Sep, 64(1), 3 - 19
Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective; Wink M; Secondary metabolites, at least the major ones present in a plant, apparently function as defence (against herbivores, microbes, viruses or competing plants) and signal compounds (to attract pollinating or seed dispersing animals) . They are thus important for the plant's survival and reproductive fitness . Secondary metabolites therefore represent adaptive characters that have been subjected to natural selection during evolution . Molecular phylogenies of the Fabaceae, Solanaceae and Lamiaceae were reconstructed and employed as a framework to map and to interpret the distribution of some major defence compounds that are typical for the respective plant families; quinolizidine alkaloids and non-protein amino acids for legumes; tropane and steroidal alkaloids for Solanaceae, and iridoids and essential oils for labiates . The distribution of the respective compounds appears to be almost mutually exclusive in the families studied, implying a strong phylogenetic and ecological component . However, on a closer look, remarkable exceptions can be observed, in that certain metabolites are absent (or present) in a given taxon, although all the neighbouring and ancestral taxa express (or do not express, respectively) the particular trait . It is argued that these patterns might reflect differential expression of the corresponding genes that have evolved earlier in plant evolution . The inconsistent secondary metabolite profiles mean that the systematic value of chemical characters becomes a matter of interpretation in the same way as traditional morphological markers . Thus, the distribution of secondary metabolites has some value for taxonomy but their occurrence apparently reflects adaptations and particular life strategies embedded in a given phylogenetic framework.

J Environ Sci (China), 2003 May, 15(3), 367 - 71
Effects of litter removal on plant species diversity: a case study in tropical Eucalyptus forest ecosystems in South China; Peng SL et al.; The removal of litterfall in the Eucalyptus plantations in South China affected the plant biodiversity in these ecosystems was found based on the field observation and lab analysis . The protection times of species diversity of three Eucalyptus communities were different (Community I with no protection, Community II with 7-year-protection, Community III with 35-year-protection) . The total numbers of species in these communities (from Community I to II to III) are 1, 6, and 17 respectively . The results showed that the protection of litterfall from being taken out of the ecosystem is important and can increase plant species diversity . This study combined biomass data, the chemical and physical properties of the soil, and the diversity of microbes in the communities . It is concluded that the mechanism of the effects of litter removal on biodiversity includes three factors: removing the suitable habitat of microbe and animal, decreasing the soil nutrient, and changing the special habitat for the germination and growth of invading plants . These results should have important implications for managing these Eucalyptus forest ecosystems in South China.

J Synchrotron Radiat, 2003 Sep 1, 10(Pt 5), 387 - 92 Epub 2003 Aug 28.
Microbeam synchrotron imaging of hairs from ancient Egyptian mummies; Bertrand L et al.; Developments in microfocus synchrotron techniques have led to new results regarding the long-term alteration of archaeological samples of biological origin . Here, ancient hair samples from two Egyptian mummies have been analyzed using a conjunction of structural and elemental synchrotron methods . In this favored context of conservation, structural analysis revealed a remarkable preservation of keratin supramolecular organization at any observed length scale . Bulk keratin structure has therefore not been modified significantly over 2000 years . However, infrared spectroscopy indicated a partial disorganization of keratins close to the hair surface through polypeptide bond breakage . Elemental mapping showed a strongly heterogeneous distribution which can be related to mummification and cosmetic treatments.

Guang Pu Xue Yu Guang Pu Fen Xi, 2002 Feb, 22(1), 126 - 30
{Studies on a sequential injection renewable surface reflectance spectrophotometric system using a microchip flow cell}; Wang JY et al.; A microchip flow cell was developed for flow injection renewable surface assay by reflectance spectrophotometry . The flow cell was coupled to a sequential injection system and optical fiber photometric detection system . The flow cell featured a three-layer structure . The flow channel was cut into a silicone rubber membrance which formed the middle layer, and a porous filter was inlayed across a widened section of the channel to trap microbeads introduced into the flow cell . The area of the detection window of the flow cell was approximately 3.6 mm2, the volume of the bead trapped in the flow cell was 2.2 microL, the depth of the bead layer was 600 microns . A multistrand bifurcated optical fiber was coupled with incident light, detector and flow cell . The chromogenic reaction of Cr(VI) with 1,5-diphenylcarbohydrazide (DPC) which was adsorbed on trapped Polysorb C-18 beads was used as a model reaction to optimize the flow cell design and the experimental system . The reflectance of the renewable reaction surface was monitored at 540 nm . With 100 microL sample loaded and 1.0 mL.min-1 carrier flow rate, the linear response range was 0-0.6 microgram.mL-1 Cr(VI) . A detection limit (3 sigma) of 6 ng.mL-1, precision of 1.5% RSD(n = 11), and a throughput of 64 samples per hour were achieved . Considerations in system and flow cell design, the influence of depth of the bead layer, weight of beads used, and the flow rates of carrier stream on the performance were discussed.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Jul;68(1 Pt 1):011706 . Epub 2003 Jul 21.
Local layer structures in circular domains of an achiral bent-core mesogen observed by x-ray microbeam diffraction; Takanishi Y et al.; The local layer structures have been investigated by x-ray microbeam diffraction in the circular domains of the SmCP phase of a banana-shaped molecule . Originally, the molecules form tilted layers with a certain tilt angle as well as nontilted ones . The application of a low electric field induces a tilted layer with a continuous change of the tilt angle; i.e., the tilted layer gradually changes the tilt angle, finally being upright at the center of circular domains . Upon application of a high electric field, the smectic layer forms a cylindrical-type structure . The layer structure changes from cylindrical to onionlike after turning off the high field.

J Immunol, 2003 Sep 1, 171(5), 2556 - 62
Intracellular mediators of granulysin-induced cell death; Okada S et al.; Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors . Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation . Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death . The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ({Ca2+}(i)), the decrease in intracellular K+, and apoptosis . Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in {Ca2+}(i) in Jurkat cells, indicating that the initial increase in {Ca2+}(i) is from intracellular stores . The rise in {Ca2+}(i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death . In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated . Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.

Scanning, 2003 Jul-Aug, 25(4), 165 - 74
Tools and procedures for quantitative microbeam isotope ratio imaging by secondary ion mass spectrometry; Gillen G et al.; In this work we demonstrate the use of secondary ion mass spectrometry (SIMS) combined with the Lispix image processing program (Bright 1995) to generate quantitative isotope ratio images from a test sample of a calcium-aluminum rich inclusion from the Allende meteorite that is known to contain discrete mineral grains with perturbed Mg isotopic ratios . Using 19.5 keV impact O- primary ion bombardment and detection of positive secondary ions, microbeam imaging SIMS has allowed us to identify, from the isotope ratio images, enrichments in the 26Mg/24Mg isotope ratio of approximately 5-15% in selected mineral grains . Using custom image processing software, each isotopic ratio image is corrected on an individual pixel basis for a number of factors including detector dead-time, mass bias effects, and isobaric interferences . We have developed procedures for correlating the isotopic images with polarized optical microscopy so that targeted mineral grains could be identified for further SIMS analysis . Finally, additional image processing tools have been developed to allow for pixel-by-pixel evaluation of the influence of detector dead-time and count rate errors on the isotopic ratio images and for correlation of the isotopic images with elemental distribution maps.

Cell Microbiol, 2003 Sep, 5(9), 581 - 92
Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation; Chamaillard M et al.; The innate immune system is the most ancestral and ubiquitous system of defence against microbial infection . The microbial sensing proteins involved in innate immunity recognize conserved and often structural components of microorganisms . One class of these pattern-recognition molecules, the Toll-like receptors (TLRs), are involved in detection of microbes in the extracellular compartment whereas a newly discovered family of proteins, the NBS-LRR proteins (for nucleotide-binding site and leucine-rich repeat), are involved in intracellular recognition of microbes and their products . NBS-LRR proteins are characterized by three structural domains: a C-terminal leucine-rich repeat (LRR) domain able to sense a microbial motif, an intermediary nucleotide binding site (NBS) essential for the oligomerization of the molecule that is necessary for the signal transduction induced by different N-terminal effector motifs, such as a pyrin domain (PYD), a caspase-activating and recruitment domain (CARD) or a baculovirus inhibitor of apoptosis protein repeat (BIR) domain . Two of these family members, Nod1 and Nod2, play a role in the regulation of pro-inflammatory pathways through NF-kappaB induced by bacterial ligands . Recently, it was shown that Nod2 recognizes a specific peptidoglycan motif from bacteria, muramyl dipeptide (MDP) . A surprising number of human genetic disorders have been linked to NBS-LRR proteins . For example, mutations in Nod2, which render the molecule insensitive to MDP and unable to induce NF-kappaB activation when stimulated, are associated with susceptibility to a chronic intestinal inflammatory disorder, Crohn's disease . Conversely, mutations in the NBS region of Nod2 induce a constitutive activation of NF-kappaB and are responsible for Blau syndrome, another auto-inflammatory disease . Nalp3, which is an NBS-LRR protein with an N-terminal Pyrin domain, is also implicated in rare auto-inflammatory disorders . In conclusion, NBS-LRR molecules appear as a new family of intracellular receptors of innate immunity able to detect specific bacterial compounds and induce inflammatory response; the dysregulation of these processes due to mutations in the genes encoding these proteins is involved in numerous auto-inflammatory disorders.

Eur J Cell Biol, 2003 Jul, 82(7), 379 - 83
Meeting report: molecular mechanisms of inflammation: how leukocytes come, see and seize; Sunderkotter C et al.; Inflammation has developed in the course of evolution as a process to defend the body against invading microbes and to respond to injuries . Several mechanisms of interaction between endothelial cells and leukocytes have evolved to render inflammation an effective, tightly controlled, and self-limited process . Imperfect executions of this "game plan" lead to pathological abnormalities resulting in diseases . The meeting on Molecular Mechanisms of Inflammation held at Schloss Elmau, Germany in October 2002 has featured activation of endothelial cells, adhesion and migration of leukocytes, as well as receptor pathways for activation and deactivation of leukocytes and, concomitantly, of the inflammatory response . Thus, a review on some of the presented data casts interesting spotlights on different steps of the inflammatory cascade.

Pathol Res Pract, 2003, 199(6), 405 - 9
Live cell catapulting and recultivation; Stich M et al.; Laser micromanipulation systems are used worldwide in the field of life science research . Most of their applications focus on the isolation of specific cells from different types of tissue and the manipulation of subcellular structures within fixed or living cells . Using the PALM MicroBeam, it is possible to microdissect living cells from a cell culture, to catapult them into collection devices, and to re-cultivate the isolated cells . For this purpose, new protocols and special equipment were developed . It has also been demonstrated that Laser Microdissection and Pressure Catapulting (LMPC) have no influence on the proliferation rate of the cells . Even re-cultivated cell colonies, trypsinized and seeded out again, are still viable after a second LMPC-procedure . This new approach opens a wide field of interesting applications in cell biology, molecular pathology, and pharmacology.

Ying Yong Sheng Tai Xue Bao, 2003 May, 14(5), 819 - 22
{Mechanisms of plant disease resistance induced by arbuscular mycorrhizal fungi}; Huang J et al.; In recent years, the induced disease resistance of plant by Arbuscular Mycorrhizal Fungi (AMF) has become a hot spot in chemo-ecological study and in biocontrol of plant disease . There were many reports indicating that AMF had antagonistic function to soilborne disease pathogen, or could suppress the growth of pathogen, and increase the resistance or tolerance of mycorrhizal plants to soilborne disease . In mycorrhizosphere, there are interactions among microbial community, in which, AMF could suppress the growth of pathogen and promote the growth of beneficial microbe . Thus, AMF may use as biocontrol fungi with other antagonism microbe . There were several hypotheses about the mechanisms of the increased resistance in mycorrhizal plants: (1) improvement of plant nutrient status; (2) competition; (3) changed roots morphology and structure; (4) changed microbial flora in rhizosphere; (5) induced resistance or systematic resistance in plant . After colonized by AMF, phenolic compounds accumulate in plant, and local defense response or systemic defense response occurs . It is necessary to do intensive research on the mechanisms of increased resistance in mycorrhizal plants, because it may help us understand the function of disease resistance of mycorrhiza correctly, and let us use AMF as a new biocontrol method to control soilborne disease in eco-agriculture in the near future.

Ying Yong Sheng Tai Xue Bao, 2003 Apr, 14(4), 618 - 22
{Advances in the research of soil microorganisms and their mediated processes under heavy metal stress}; Yu S et al.; More and more evidence from both laboratory and field experiments suggested that soil microbes were more sensitive to heavy metal stress than soil animals or plants living in the same soil . This understanding has been gradually accepted as a common point in this field . The history and the latest advances in soil microorganisms and its mediated processes under heavy metal stresses in soils were reviewed in this paper . It is difficult to do any comparison among the results from laboratory, field toxicological experiment and environmental monitoring . So far, a relevantly 'accurate' control soil was rarely available in the environmental monitoring . Much of evidence has indicated that not only microbial population/biomass was changed under heavy metal stress, but also its structure was varied . Although many molecular methods, such as PLFA, BIOLOG and DNA, have been developed to detect this microbial structural change, they always need many expensive instruments and accessories, and only could be done in laboratory . Therefore, some rapid and easy methods are expected to develop to substitute the traditional plate count . The speciation in the solution phase, ion effect and rhizospheric effect of heavy metal on the toxicity to soil microorganisms and its mediated processes need extent studies and discussion . Finally, the microbial indicator system of assessing heavy metal toxicity to soil microorganisms and its mediated processes need to be established.

Biomol Eng, 2003 Jul, 20(4-6), 291 - 8
A marine biological underwater depuration system (MUDS) to process waste waters; Cattaneo-Vietti R et al.; An underwater device, able to favour the sea auto-cleaning capacities, is herein described . This system, called MUDS (marine underwater depuration system), consists of a percolating filter and is placed at sea over an urban sewage outflow of a submarine pipeline . Due to the density difference, the water effluent flows through the percolating filter: this favours the mixing and a prompt recycling of organic matter, activating a marine trophic web . Rich microbenthic communities develop on the MUDS, both interstitially, inside the filter, and on the structure . The community mainly consists of ciliates, nematodes, harpacticoid copepods and polychaetes, all of which being organisms that increase the depuration efficiency by consumption of organic matter . This structure acts also as a deterrent for the illegal trawling activity in the area, and attracts large numbers of several finfish species, thus working as a fish aggregating device (FAD) . It is possible to utilise this underwater device for medium littoral towns with strong differences in effluent discharges during the year, where the use of land-built effluent treatment plants is too expensive.

AAPS PharmSciTech . 2002;3(3):E25.
Preparation of gelatin microbeads with a narrow size distribution using microchannel emulsification; Iwamoto S et al.; The purpose of this study was to prepare monodisperse gelatin microcapsules containing an active agent using microchannel (MC) emulsification, a novel technique for preparing water-in-oil (W/O) and oil-in-water (O/W) emulsions . As the first step in applying MC emulsification to the preparation of monodisperse gelatin microcapsules, simple gelatin microbeads were prepared using this technique . A W/O emulsion with a narrow size distribution containing gelatin in the aqueous phase was created as follows . First, the aqueous disperse phase was fed into the continuous phase through the MCs at 40 degrees C (operating pressure: 3.9 kPa) . The emulsion droplets had an average particle diameter of 40.7 microm and a relative standard deviation of 5.1% . The temperature of the collected emulsion was reduced and maintained at 25 degrees C overnight . The gelatin microbeads had a smooth surface after overnight gelation; the average particle diameter was calculated to be 31.6 microm, and the relative standard deviation, 7.3% . The temperature was then lowered to 5 degrees C by rapid air cooling and finally dried . The gelatin beads were dried and could be resuspended well in iso-octane . They had an average particle diameter of 15.6 microm, and a relative standard deviation of 5.9% . Using MC emulsification, we were able to prepare gelatin microbeads with a narrow size distribution . Since this emulsification technique requires only a low-energy input, it may create desirable experimental conditions for microencapsulation of unstable substances such as peptides and proteins . This method is promising for making monodisperse microbeads.

Hist Cienc Saude Manguinhos, 2002, 9 Suppl, 111 - 38
{The tropics, microbes, and vectors}; Caponi S; The article analyzes how Argentinean and Brazilian researchers designed their tropical disease research programs in the late 19th and early 20th centuries . Even though Brazilian and Argentinean hygiene were direct descendants of Pasteurian programs and principles, Brazil addressed its public health problems through a new research agenda that embraced bacteriology, parasitology, and a concern with vectors . Argentina, on the other hand, disregarded the problems caused by tropical disease, insisting instead on reducing all of its health issues to those that could be approached through a paradigm embracing microbiology studies and the "aerista" strategies typical of classic hygiene.

BMC Biotechnol . 2003 Aug 12;3(1):13.
Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse; Wang W et al.; BACKGROUND: cDNA microarrays have the potential to identify the genes involved in invasion and metastasis . However, when used with whole tumor tissue, the results average the expression patterns of different cell types . We have combined chemotaxis-based cell collection of the invasive subpopulation of cells within the primary tumor with array-based gene expression analysis to identify the genes necessary for the process of carcinoma cell invasion . RESULTS: Invasive cells were collected from live primary tumors using microneedles containing chemotactic growth factors to mimic chemotactic signals thought to be present in the primary tumor . When used with mammary tumors of rats and mice, carcinoma cells and macrophages constitute the invasive cell population . Microbeads conjugated with monoclonal anti-CD11b (Mac-1alpha) antibodies were used to separate macrophages from carcinoma cells . We utilized PCR-based cDNA amplification from small number of cells and compared it to the quality and complexity of conventionally generated cDNA to determine if amplified cDNA could be used with fidelity for array analysis of this cell population . These techniques showed a very high level of correlation indicating that the PCR based amplification technique yields a cDNA population that resembles, with high fidelity, the original template population present in the small number of cells used to prepare the cDNA for use with the chip . CONCLUSIONS: The specific collection of invasive cells from a primary tumor and the analysis of gene expression in these cells are is now possible . By further comparing the gene expression patterns of cells collected by invasion into microneedles with that of carcinoma cells obtained from the whole primary tumor, the blood, and whole metastatic tumors, genes that contribute to the invasive process in carcinoma cells may be identified.

Pediatr Allergy Immunol, 2003 Aug, 14(4), 255 - 60
The C-159T polymorphism in the CD14 promoter is associated with serum total IgE concentration in atopic Chinese children; Leung TF et al.; Activation of macrophages through CD14 by microbes is crucial in inducing immunity by type 1 T helper cells . A C-to-T polymorphism at position -159 of CD14 was associated with serum total IgE level in Caucasians but not in Japanese subjects . The objective of this study is to determine whether this polymorphic marker is associated with atopy and asthma phenotypes in Chinese children . Restriction fragment length polymorphism was used to characterize CD14/-159 genotypes . Microparticle immunoassay was used to measure serum total IgE level; fluorescent enzyme immunoassay was performed to measure serum concentrations of specific IgE to aeroallergens; and enzyme-linked immunosorbent assay was used to measure serum levels of soluble CD14 (sCD14) . Lung function in asthmatics was assessed by spirometry . Two hundred and fifty-eight patients and 92 control children were recruited . Their mean serum total IgE concentrations were 331 and 74 kIU/l, respectively (p < 0.0001) . Atopy, defined as the presence of at least one allergen-specific IgE in serum, was found in 220 (85%) patients and in 41 (45%) controls (p < 0.0001) . Serum sCD14 levels were significantly associated with CD14/-159 genotypes (p = 0.004) . Atopic subjects with CC genotype in CD14/-159 had the highest serum total IgE levels compared with CT and TT genotypes, with the respective mean values being 661, 427 and 380 kIU/l (p = 0.015) . Similarly, a higher proportion of subjects with CC genotype had increased serum total IgE concentration (p = 0.039) . This polymorphic marker was not associated with asthma or aeroallergen sensitization in our cohort . Our results suggest that the C-159T of CD14 was associated with serum total IgE concentration in atopic Chinese children.

Appl Biochem Biotechnol, 2003 Jul, 110(1), 1 - 10
Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane: comparison of enzyme performances with inorganic beaded supports; Leiriao PR et al.; A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 micromol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) . Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde . The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8 U/mg of protein) . Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 micromol of H2O2 reduced/(h x Uimmob HRP).

J Microencapsul, 2003 Sep-Oct, 20(5), 597 - 611
Influence of alginate characteristics on the properties of multi-component microcapsules; Wandrey C et al.; A variety of sodium alginates, differing in molar mass and structural composition, have been evaluated in the preparation of multi-component microbeads and microcapsules . Bead formation occurred by gelation with calcium chloride . Capsules were produced by reacting the pre-formed beads with the oligocation poly(methylene-co-guanidine) . Despite the equiponderous (1:1) mixing with a second polyanion, sodium cellulose sulphate, the influence of the alginate properties remains evident . Specifically, the effect of the chemical composition was found to be more significant than that of the molar mass for both the mechanical and transport properties . Furthermore, for alginates of 73% alpha-l-guluronic acid content less shrinking was observed compared to the 38% guluronic materials . This results in the case of the same encapsulator settings in larger microsphere diameters and thicker membranes accompanied by enhanced mechanical resistance though, also, in a higher permeability for the high-G capsules . However, subsequent coating with lower molar mass alginate allows one to adjust the permeability over a broad range, suitable for cell encapsulation and immunoprotection, without compromising the durability.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Oct, 24(5), 495 - 500
{Comparison of capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control}; Xiao J et al.; OBJECTIVE: To explore in vitro expansion of CD34+CD59+ cells from patients with PNH, and compare the capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control . METHODS: CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control were selected from the bone marrow mononuclear cells by means of two-step sorting method with immunomagnetic microbead-flow cytometry, then underwent in vitro expansion for two weeks and semi-solid culture in vitro before and after expansion . RESULTS: (1) CD34+CD59+ cells from patients with PNH can be expanded effectively in vitro, and the biggest expansion of CD34+CD59+ cells was about 23.49 fold on the 7th day . (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, such as: the best combination of hematopoietic factors for in vitro expansion was SCF+ IL-3 + IL-6 + FL + Tpo + Epo, and the seventh day was the most suitable in course of 4-14 days for in vitro expansion, and after in vitro expansion, the cells remained CD59 positive and strong capability of performing colony-forming . (3) CD34+ cells from normal control had better proliferation, expansion and stronger potential to survive than CD34+CD59+ cells from patients with PNH . CONCLUSIONS: (1) In vitro expansion of CD34+CD59+ cells from patients with PNH can be performed . The present study showed the possibility of performing ABMT or APBSCT clinically for patients with PNH . (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, but the latter had better proliferation, expansion and stronger potential to survive than the former . CD34+CD59+ cells from patients with PNH were not completely normal cells.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Aug, 24(4), 433 - 6
{Toll-like receptor and innate immunity in phylogenesis}; Liu Y et al.; In protection against microbes, an organism recognizes the pathogen associated molecular pattern (PAMP) on microbes by pattern recognition receptor (PRR) . Toll-like receptor is called innate immunity . A family of cell membrane receptor was found in recent years that can mediate innate immune responses through the activation of a series of immune-related genes . In phylogenesis, it is highly conservative . However, its functions are getting more diversified with the complication of the immune functions of organisms.

Oecologia, 2003 Nov, 137(3), 399 - 404 Epub 2003 Aug 07.
Mycorrhizal colonization mediated by species interactions in arctic tundra; Urcelay C et al.; The Alaskan tussock tundra is a strongly nutrient-limited ecosystem, where almost all vascular plant species are mycorrhizal . We established a long-term removal experiment to document effects of arctic plant species on ecto- and ericoid mycorrhizal fungi and to investigate whether species interactions and/or nutrient availability affect mycorrhizal colonization . The treatments applied were removal of Betula nana ( Betulaceae, dominant deciduous shrub species), removal of Ledum palustre ( Ericaceae, dominant evergreen shrub species), control (no removal), and each of these three treatments with the addition of fertilizer . After 3 years of Ledum removal and fertilization, we found that overall ectomycorrhizal colonization in Betula was significantly reduced . Changes in ectomycorrhizal morphotype composition in removal and fertilized treatments were also observed . These results suggest that the effect of Ledum on Betula's mycorrhizal roots is due to sequestration of nutrients by Ledum, leading to reduced nutrient availability in the soil . In contrast, ericoid mycorrhizal colonization was not affected by fertilization, but the removal of Betula and to a lower degree of Ledum resulted in a reduction of ericoid mycorrhizal colonization suggesting a direct effect of these species on ericoid mycorrhizal colonization . Nutrient availability was only higher in fertilized treatments, but caution should be taken with the interpretation of these data as soil microbes may effectively compete with the ion exchange resins for the nutrients released by plant removal in these nutrient-limited soils.

Microb Ecol, 2003 Nov, 46(4), 442 - 53 Epub 2003 Aug 14.
Dynamics of microbial populations and strong selection for Cycloclasticus pugetii following the Nakhodka oil spill; Maruyama A et al.; Microbial population changes were monitored immediately after the Nakhodka oil spill accident in January 1997 at the heavily oil-contaminated Mikuni coast along the Sea of Japan . The total cell number was almost stable for one year at 2-5 x 10(5) cells mL(-1), while the relative occurrence of culturable heterotrophs and degraders of oil components such as C-heavy oil, kerosene, and n-tetradecane varied, showing a maximum (>50% of the total) immediately following the accident . Gene amplification and phylogenetic analysis of a dilution culture using C-heavy oil as the sole carbon and energy source revealed that one of the predominant oil degraders at the oil-contaminated coast in 2 weeks after the accident closely resembled the aromatic hydrocarbon decomposer Cycloclasticus pugetii . Microbial community composition in oil-contaminated seawater was estimated at the molecular level using newly developed oligonucleotide probes, probe wash-off curve estimation, and quantitative fluorescence dot-blot hybridization techniques . At two different oil-polluted sites, harbor and intertidal regions, the C . pugetii group was estimated to make up 23-25% of the total Bacteria population, followed by the aliphatic hydrocarbon decomposer Alcanivorax borkumensis, which formed 4-7% of the Bacteria . In incubation experiments using floated oil slick and indigenous microbes collected at the harbor, oil degradation activities were enhanced by the addition of both organic and inorganic nutrients . Significant decreases were found in aromatic and aliphatic hydrocarbon fractions: 54-60% and 22-24% in 2 weeks to 68-77% and 23-32% in 2 months, respectively.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 10124 - 8 Epub 2003 Aug 06.
The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense; Di Matteo A et al.; Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that protect plants from fungal invasion . They interact with endopolygalacturonases secreted by phytopathogenic fungi, inhibit their enzymatic activity, and favor the accumulation of oligogalacturonides, which activate plant defense responses . PGIPs are members of the leucine-rich repeat (LRR) protein family that in plants play crucial roles in development, defense against pathogens, and recognition of beneficial microbes . Here we report the crystal structure at 1.7-A resolution of a PGIP from Phaseolus vulgaris . The structure is characterized by the presence of two beta-sheets instead of the single one originally predicted by modeling studies . The structure also reveals a negatively charged surface on the LRR concave face, likely involved in binding polygalacturonases . The structural information on PGIP provides a basis for designing more efficient inhibitors for plant protection.

Appl Environ Microbiol, 2003 Aug, 69(8), 4927 - 34
Metagenomic profiling: microarray analysis of an environmental genomic library; Sebat JL et al.; Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms . However, conventional library-screening techniques permit characterization of relatively few environmental clones . Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities . A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls . COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species . The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe . Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes . Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture . Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition . Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

J Theor Biol, 2003 Sep 7, 224(1), 97 - 105
Exact solutions to the continuous-quality equation for soil organic matter turnover; Bosatta E et al.; All living systems depend on transformations of elements between different states . In particular, the transformation of dead organic matter in the soil (SOM) by decomposers (microbes) releases elements incorporated in SOM and makes the elements available anew to plants . A major problem in analysing and describing this process is that SOM, as the result of the decomposer activity, is a mixture of a very large number of molecules with widely differing chemical and physical properties . The continuous-quality equation (CQE) is a general equation describing this complexity by assigning a continuous-quality variable to each carbon atom in SOM . The use of CQE has been impeded by its complicated mathematics . Here, we show by deriving exact solutions that, at least for some specific cases, there exist solutions to CQE . These exact solutions show that previous approximations have overestimated the rate by which litter decomposes and as a consequence underestimated steady state SOM amounts . The exact and approximate solutions also differ with respect to the parameter space in which they yield finite steady-state SOM amounts . The latter point is important because temperature is one of the parameters and climatic change may move the solution from a region of the parameter space with infinite steady-state SOM to a region of finite steady-state SOM, with potentially large changes in soil carbon stores . We also show that the solution satisfies the Chapman-Kolmogorov theorem . The importance of this is that it provides efficient algorithms for numerical solutions.

FEMS Immunol Med Microbiol, 2003 Aug 18, 38(1), 9 - 12
Immunobiotics and the probiotic evolution; Clancy R; The term 'probiotic' was coined by the food industry to describe microbes beneficial to health . Scientific criticism based on poorly characterised isolates, soft clinical targets, and an absence of a mechanistic framework limited value of this concept . Current clinical and animal model studies support a role for certain 'probiotics', activating the common mucosal system through the stimulation of gut antigen-presenting cells to both promote protection and to switch regulatory mechanisms . It is concluded that a new term is required to identify bacteria that promote health through driving mucosal immune mechanisms, compared to those with strictly local effects . The term 'immunobiotics' is suggested as appropriate to fulfil this need.

Prostaglandins Leukot Essent Fatty Acids, 2003 Aug-Sep, 69(2-3), 195 - 205
The role of mast cells in asthma; Boyce JA; While the role of mast cells in allergic reactions is unequivocal, their precise functions in asthma remain controversial . Mast cells uniquely populate all vascularized organs and tissues, including the upper and lower respiratory tree, even in healthy individuals . Histologic evidence suggests that asthma is accompanied by a mast cell hyperplasia in the inflamed mucosal epithelium and the adjacent smooth muscle . The mechanisms responsible for constitutive mast cell development have been partly elucidated . Moreover, both in vitro studies and in vivo disease models indicate that mast cells have a remarkably flexible program of gene expression, and this program can be drastically altered by the T-cell-derived Th2 cytokines relevant to asthma . Moreover, the role of mast cells in innate immunity is now firmly established, and the capacity for numerous microbial pathogens to initiate their activation in vitro and in vivo suggest mechanisms by which microbes could initiate disease exacerbations.

Exp Neurol, 2003 Aug, 182(2), 373 - 82
Neural trans-differentiation potential of hepatic oval cells in the neonatal mouse brain; Deng J et al.; Although the existence of the hepatic oval cell (HOC), the liver stem cell has been known for almost 70 years, little is known about the potential for this adult stem cell to trans-differentiate into cells of other tissues . While their origin remains enigmatic, HOCs share many similarities with hematopoietic stem cells . Recent studies have revealed that a small percentage of HOCs can arise from a bone marrow-derived stem cell source . Here we report that, like bone marrow stem cells, HOCs can survive transplantation to the neonatal mouse brain and show signs of trans-differentiation by adopting the morphology and antigenic phenotype of both macro- and microglia cells . Trans-differentiated microglia cells were functional, showing active phagocytosis when cotransplanted with latex microbeads in vivo . In addition to glial markers, a small number of transplanted HOCs were immunopositive for neuronal markers, but displayed ambiguous phenotype, making their characterization difficult.

Mol Pathol, 2003 Aug, 56(4), 240 - 3
Improved resolution by mounting of tissue sections for laser microdissection; van Dijk MC et al.; BACKGROUND: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections . However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor . AIMS: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated . METHODS: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue . The mounting solution consisted of an adhesive gum and blue ink diluted in water . Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue . RESULTS: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA . One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser . To minimise this effect, the mounting solution should be diluted as much as possible . Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser . CONCLUSIONS: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

FASEB J, 2003 Aug, 17(11), 1422 - 7
Bystander effect induced by counted high-LET particles in confluent human fibroblasts: a mechanistic study; Shao C et al.; The possible mechanism of a radiation-induced bystander response was investigated by using a high-LET heavy particle microbeam, which allows selected cells to be individually hit with precise numbered particles . Even when only a single cell within the confluent culture was hit by one particle of 40Ar (approximately 1260 keV/microm) or 20Ne (approximately 380 keV/microm), a 1.4-fold increase of micronuclei (MN) was detected demonstrating a bystander response . When the number of targeted cells increased, the number of MN biphasically increased; however, the efficiency of MN induction per targeted cell markedly decreased . When 49 cells in the culture were individually hit by 1 to 4 particles, the production of MN in the irradiated cultures were approximately 2-fold higher than control levels but independent of the number and LET of the particles . MN induction in the irradiated-culture was partly reduced by treatment with DMSO, a scavenger of reactive oxygen species (ROS), and was almost fully suppressed by the mixture of DMSO and PMA, an inhibitor of gap junctional intercellular communication (GJIC) . Accordingly, both ROS and GJIC contribute to the above-mentioned bystander response and GJIC may play an essential role by mediating the release of soluble biochemical factors from targeted cells.

Med Hypotheses, 2003 Aug, 61(2), 235 - 9
Crossroads of extrathymic lymphocytes maturation pathways; Bubanovic IV; The majority of T cells located in peripheral lymphoid organs are dependents on the thymus for regular differentiation and function . Only a minority of T lymphocytes are thymus-independent . These cells pass by extrathymic maturation processes and become mature T lymphocytes . Some data suggest that mechanism of extrathymic lymphocytes maturation (eTLM) includes migration, proliferation, differentiation and selection of lymphocytes as well as thymic pathway . With aging and progression of thymic involution or in accidental thymic involution, pathway of eTLM derives emphasis . T cells from extrathymic pathway probably can polarize action of thymic-dependent T cells or participate in immune reaction in antigen-destructive or antigen-protective manners . Consequently, extrathymic pathways can be a source of self-reactive T cells or cells which participate in mechanisms of trophoblast or tumor escape . Results of eTLM probably are not presets, already depend upon many factors and microenvironmental snapshots . Factors like cytokines, prostaglandine, microbes, MHC molecules, hormones, Fas ligand, heat shock proteins, phenotypes of dendritic cells and APCs, probably can be polarizing courses of eTLM pathway . Definitive to the course of extrathymic-derived cells action, presumably is resultant of microenvironmental relations and interactions of foregoing factors . Hypothesis that microbes, especially viruses, can be promoters of extrathymic (self)antigen-reactive lymphocytes maturation is real as well as hypothesis that extrathymic lymphocytes selection and products of selected lymphocytes can be included in mechanisms of tumor, trophoblast and transplant rejection or escape.

Med Hypotheses, 2003 Aug, 61(2), 182 - 9
A metabolic basis for fibromyalgia and its related disorders: the possible role of resistance to thyroid hormone; Garrison RL et al.; It has long been recognized that the symptom complex of fibromyalgia can be seen with hypothyroidism . Hypothyroidism may been categorized, like diabetes, into type I (hormone deficient) and type II (hormone resistant) . Most cases of fibromyalgia fall into the latter category . The syndrome is reversible with treatment, and is usually of late onset . It is likely more often acquired than due to mutated receptors . Now that there is evidence to support the hypothesis that fibromyalgia may be due to thyroid hormone resistance, four major questions appear addressable . First, can a simple biomarker be found to help diagnose it? Second, what other syndromes similar to Fibromyalgia may share a thyroid-resistant nature? Third, in non-genetic cases, how is resistance acquired? Fourth, what other methods of treatment become available through this new understanding? Preliminary evidence suggests that serum hyaluronic acid is a simple, inexpensive, sensitive, and specific test that identifies fibromyalgia . Overlapping symptom complexes suggest that chronic fatigue syndrome, Gulf war syndrome, premenstrual syndrome, post traumatic stress disorder, breast implant silicone sensitivity syndrome, bipolar affective disorder, systemic candidiasis, myofascial pain syndrome, and idiopathic environmental intolerance are similar enough to fibromyalgia to merit investigation for possible thyroid resistance . Acquired resistance may be due most often to a recently recognized chronic consumptive coagulopathy, which itself may be most often associated with chronic infections with mycoplasmids and related microbes or parasites . Other precipitants of thyroid resistance may use this or other paths as well . In addition to experimentally proven treatment with supraphysiologic doses of thyroid hormone, the thyroid-resistant disorders might be treatable with anti-hypercoagulant, anti-infective, insulin-sensitizing, and hyaluronolytic strategies.

Neuropathol Appl Neurobiol, 2003 Aug, 29(4), 325 - 40
The pathology and pathogenesis of retinal vasculitis; Hughes EH et al.; Retinal vasculitis is a rare, but potentially blinding intraocular inflammatory condition with diverse aetiology . Although commonly idiopathic, it has a strong association with systemic inflammatory diseases known to involve other areas of the central nervous system, most notably Behcet's disease, sarcoidosis, systemic lupus erythematosis and multiple sclerosis . This article describes the clinicopathologic features of retinal vasculitis and its visually damaging sequelae, reviewing available human histopathologic studies and work with experimental models to discuss the pathogenesis and immunopathology . Evidence indicates that noninfective retinal vasculitis is an autoimmune condition that may be induced by antecedent infection with microbes cross-reacting with putative autoantigens, influenced by genetic susceptibility of both HLA associations and cytokine polymorphisms . The growing understanding of the cellular mechanisms involved in the effector immune response is already providing a rationale for more specific therapeutic approaches.

Phys Med Biol, 2003 Jul 7, 48(13), R1 - 35
Medical applications of synchrotron radiation; Suortti P et al.; The medical imaging and therapeutic technologies that are based on the use of radiation are reviewed briefly, with special emphasis on the recent developments of synchrotron radiation (SR) methods . New results have been achieved in all of these areas since the last comprehensive reviews were written in this field . This topical review is intended to make the latest possible results and complete set of references available . The different contrast mechanisms in imaging by x-rays are described . The applications range from whole-body imaging to studies of atomic and molecular structures . The SR imaging applications include coronary angiography, bronchography, mammography, computed tomography, x-ray microscopy and imaging by scattering . The therapy applications include photon activation therapy and microbeam radiation therapy.

Bull Math Biol, 2003 Jul, 65(4), 747 - 66
Probabilistic modelling for estimating gas kinetics and decompression sickness risk in pigs during H2 biochemical decompression; Fahlman A et al.; We modelled the kinetics of H2 flux during gas uptake and elimination in conscious pigs exposed to hyperbaric H2 . The model used a physiological description of gas flux fitted to the observed decompression sickness (DCS) incidence in two groups of pigs: untreated controls, and animals that had received intestinal injections of H2-metabolizing microbes that biochemically eliminated some of the H2 stored in the pigs' tissues . To analyse H2 flux during gas uptake, animals were compressed in a dry chamber to 24 atm (ca 88% H2, 9% He, 2% O2, 1% N2) for 30-1440 min and decompressed at 0.9 atm min(-1) (n = 70) . To analyse H2 flux during gas elimination, animals were compressed to 24 atm for 3 h and decompressed at 0.45-1.8 atm min(-1) (n = 58) . Animals were closely monitored for 1 h post-decompression for signs of DCS . Probabilistic modelling was used to estimate that the exponential time constant during H2 uptake (tau(in)) and H2 elimination (tau(out)) were 79 +/- 25 min and 0.76 +/- 0.14 min, respectively . Thus, the gas kinetics affecting DCS risk appeared to be substantially faster for elimination than uptake, which is contrary to customary assumptions of gas uptake and elimination kinetic symmetry . We discuss the possible reasons for this asymmetry, and why absolute values of H2 kinetics cannot be obtained with this approach.

Nature, 2003 Aug 14, 424(6950), 743 - 8 Epub 2003 Jul 20.
Identification of Lps2 as a key transducer of MyD88-independent TIR signalling; Hoebe K et al.; In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response . Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer . Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1 . Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected . Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor . However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS . This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.

Can J Ophthalmol, 2003 Jun, 38(4), 285 - 8
Soft-tissue fillers for wrinkles, folds and volume augmentation; Jordan DR; Hyaluronic acid gels, with or without dextran beads, and collagen with suspended PMMA microbeads are exciting new soft-tissue fillers that are becoming increasing popular for facial rejuvenation . Each material provides wrinkle reduction, contour improvement and volume augmentation when placed into lines and furrows or when used for lip augmentation . The duration of action varies from 6 months to permanent, depending on the product.

Curr Opin Mol Ther, 2003 Jun, 5(3), 235 - 40
Analysis of RNA-protein interactions by flow cytometry; Brodsky AS et al.; Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions . Applications of this technology include antibody assays and single nucleotide polymorphism mapping . This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes . The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.

Transfusion, 2003 Aug, 43(8), 1029 - 37
West Nile virus in blood: stability, distribution, and susceptibility to PEN110 inactivation; Mather T et al.; BACKGROUND: The outbreak of West Nile virus (WNV) is the most recent reminder that the blood supply continues to be vulnerable to emerging and reemerging pathogens . A potentially prospective approach to reducing the risk of transfusion-transmitted infections of a known or newly emerging microbe is implementation of a broad-spectrum pathogen reduction technology . The purpose of this study was to evaluate the susceptibility of WNV to PEN110 inactivation in RBCs and to characterize the WNV interaction with blood, including the stability of WNV in RBCs stored at 1 to 6 degrees C, its distribution and infectivity, and its ability to infect WBCs . STUDY DESIGN AND METHODS: Inactivation was performed with three WNV isolates spiked into WBC-reduced RBCs . The stability of the virus was evaluated by spiking two viral loads into RBCs followed by storing at 1 to 6 degrees C for up to 42 days . The distribution of the virus in plasma, RBCs, and PBMCs was evaluated with whole blood from infected hamsters . Finally, in vitro propagation of WNV was evaluated with the THP-1 cell line and primary monocytes . RESULTS: The kinetics of PEN110 inactivation of WNV isolates RI-44, NJ-176, and 99-3494031 were fast and complete within 24 hours with reduction factors of 5 to 7 log plaque-forming units per mL . WNV remained infectious for up to 42 days at 1 to 6 degrees C . The WNV titers in whole blood, plasma, RBCs, and PBMC fractions were equally distributed and ranged from 2 to 3 log tissue culture infectious dose 50 percent per mL . Productive infection of stimulated monocytes and THP-1 cells was also demonstrated . CONCLUSIONS: These studies demonstrated that PEN110 efficiently inactivated WNV in RBCs and whole blood from infected hamsters to the limit of detection . WNV survived in RBCs stored at 1 to 6 degrees C with a gradual loss of titer but infectivity could still be observed for up to 42 days . In addition, it was observed that WNV was equally distributed in all blood fractions including PBMCs and it was possible to establish productive infection of a human monocytic cell line and stimulated human monocytes.

Analyst, 2003 Jun, 128(6), 547 - 51
Isolation and extraction of target microbes using thermal sol-gel transformation; Arai F et al.; We developed a novel separation method for random screening of target microorganisms from a large heterogeneous population by using a local viscosity control . A thermal sol-gel transformation material is mixed with the sample liquid and we controlled the state from sol to gel and gel to sol reversibly based on the temperature change controlled by heating of the microelectrode with the electric current and focused laser irradiation near the target . The selected microorganisms are fixed on the bottom plate by gel, since the viscosity around the target is temporally increased by the local heating by the focused laser . The other objects are easily washed away by the cleaning flow in the microchamber . Process of fixation, cleaning, isolation and extraction of the target microbe was possible in very short time . Based on this method, two separation systems are developed and basic experimental results of fixation and isolation of targets are shown.

Zhonghua Xue Ye Xue Za Zhi, 2003 Apr, 24(4), 169 - 73
{Study of biological characteristics of the CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients' bone marrow}; Xiao J et al.; OBJECTIVE: To explore the characteristics of CD(34)(+) CD(59)(+) cells from paroxysmal nocturnal hemoglobinuria(PNH) patients' bone marrow and the possible reasons of hematopoietic clonal dominance of PNH clones . METHODS: CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) cells from normal control were selected from the bone marrow mononuclear cells by means of immunomagnetic microbead-flow cytometry two step sorting method undergone ex vivo expansion in liquid culture for two weeks and performed semisolid cultures before and after expansion . RESULTS: (1) Cultivation for seven days was the optimum for ex vivo expansion of PNH CD(34)(+) CD(59)(+) cells and normal CD(34)(+) cells, both cell populations remained CD(59) positive after expansion . (2) Normal CD(34)(+) cells had higher capacities of proliferation and expansion, and stronger potential to survival than that of both PNH CD(34)(+) CD(59)(+) and PHN CD(34)(+) CD(59)(-) cells . (3) In terms of semisolid culture, there was no significant difference in the yields of CFU formation between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells . (4) In liquid culture with combinations of hematopoietic factors SCF + IL-3 + IL-6 + FL + Tpo or SCF + IL-3 + IL-6 + FL + Tpo + Epo, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells; but with combination of SCF + IL-3 + IL-6 + FL + Tpo + Epo + GM-CSF, CD(34)(+) CD(59)(-) cells had better proliferation and expansion capacities and stronger potential to survival than that of CD(34)(+) CD(59)(+) cells . CONCLUSIONS: (1) Normal CD(34)(+) cells had better proliferation, expansion capacities and stronger potential to survival than that of PNH CD(34)(+) CD(59)(+)cells . (2) In semisolid and liquid culture with hematopoietic factor combinations, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells . It was suggested that CD(34)(+) CD(59)(-) cells had no clonal hemotopoiesis dominance . GM-CSF might be one of the reasons for PHN clones to possess clonal hematopoiesis dominance.

Cell Microbiol, 2003 Aug, 5(8), 493 - 500
Dendritic cells and host resistance to infection; Moll H; Host defence against infection requires an integrated response of both the innate and adaptive arms of the immune system . Emerging data indicate that dendritic cells contribute an essential part to the initiation and regulation of adaptive immunity . Dendritic cells guard the sites of pathogen entry to the host and are uniquely suited to detect and capture invading microbes . Upon recognition of microbial structures and appropriate activation, a maturation programme is triggered and dendritic cells migrate to lymphoid organs to stimulate a primary cell-mediated immune response . Moreover, dendritic cells play a critical role in shaping the emerging response, thereby controlling the course of infection . They can discriminate between various types of microorganisms and are capable of producing different cytokines in response to different microbial stimuli . On the other hand, pathogens developed numerous strategies to evade and subvert dendritic cell functions . Elucidating the interactions of dendritic cells with microbial pathogens may lead to novel strategies for combating infectious diseases by dendritic cell-based vaccination and immunotherapy . This review highlights recent advances in our knowledge of the unique role of dendritic cells in counteracting microbial infections.

Eur J Clin Invest, 2003 Aug, 33(8), 657 - 61
Do temporal arteritis lesions contain bacterial DNA?
Renko J, Kalela A, Karhunen PJ, Helin H, Sillanaukee P, Nikkari S, Nikkari ST.
BACKGROUND: Temporal arteritis is a primary vascular inflammatory disease . The aetiology of temporal arteritis is unknown, but the influence of environmental factors such as infections has been suggested . MATERIALS AND METHODS: We used broad-range PCR, targeting conserved regions of the gene encoding for ribosomal RNA, to detect bacterial DNA in 27 temporal artery biopsies . Five uninvolved temporal arteries were also included . A lung sample of confirmed bacterial pneumonia served as a positive control . Inflammation was examined by histochemistry and light microscopy . RESULTS: The sensitivity of the broad-range PCR assay was 5.0 fg of DNA . Bacterial DNA sequences were neither detected in 27 temporal arteritis specimens nor in the normal temporal artery samples . However, bacterial DNA was successfully amplified from the lung sample of a subject with pneumonia . In addition, human DNA was amplified by primers for human beta-actin from all clinical specimens, suggesting lack of significant inhibitors of the molecular amplification reaction . Histochemistry showed signs of strong inflammation in the arteritis samples . CONCLUSIONS: The lack of detectable amounts of bacterial DNA suggests that viable bacteria do not have a role in chronic stages of temporal arteritis . However, these findings do not rule out the possibility of bacterial degradation products as stimulants of chronic inflammation, or of viable microbes as triggering factors of acute temporal arteritis.

Ann N Y Acad Sci, 2003 Jun, 990, 743 - 50
Probabilistic evaluation of aspirating doses of a Q fever pathogen; Vorobeychikov EV et al.; The process of biological aerosol penetration into respiratory organs is connected with the estimation of the amount of an aspiration dose of microorganisms (D) a human gets in the center of infection . Here we submit a probabilistic model describing the process of hits of a Q fever pathogen in a human respiratory tract . This approach makes it possible to get qualitatively different probabilistic estimations of doses received and corresponding values of critical time intervals of exposure at which the amount accumulated in the respiratory tract turns into an infecting dose . This permits one to approach the explanation of the infection process in the context of the absence of its absolute nature, which is connected both with the immune system responses and the irregularity with which the recipient receives aspiration doses under conditions of artificial distribution of the microbe-bearing aerosol.

Radiat Res, 2003 Aug, 160(2), 133 - 42
Response of rat skin to high-dose unidirectional x-ray microbeams: a histological study; Zhong N et al.; There is growing interest in evaluating microbeam radiation therapy as a potential clinical modality . Microbeam radiation therapy uses arrays of parallel, microscopically thin (<100 microm) planes of synchrotron-generated X rays (microplanar beams, or microbeams) . Due to the relatively low beam energies involved in microbeam radiation therapy (a median beam energy of 120 keV was used in the present study), the dose penetration of microbeams in tissue is lower than that used in conventional radiotherapy . This lower energy necessitates using a significantly elevated dose to the skin's surface during clinical microbeam therapy to ensure an adequate dose distribution in the target tumor . The findings of the present study, using a rat skin model, indicated that the skin had an extremely high tolerance to microbeam radiation at doses considerably in excess of those that were therapeutically effective in preclinical studies . A histological study was undertaken to evaluate the biological mechanisms underlying this high tolerance . The irradiation configuration employed single-exposure, unidirectional microbeams 90 microm wide, with 300 microm beam spacing on-center . The in-beam skin-surface absorbed doses were in the range 835-1335 Gy . Monte Carlo simulations of the dose distribution indicated that the "valley" dose, i.e . the radiation leakage between adjacent microbeams, was about 2.5% of the in-beam dose . The high tolerance of the rats' skin to microbeams and the rapid regeneration of the damaged segments of skin were attributed to the surviving clonogenic cells situated between the adjacent microplanar beams . In the epidermis, clonogenic cells in the hair follicular epithelium appeared to play a key role in the regeneration process.

J Cosmet Sci, 2003 May-Jun, 54(3), 239 - 50
Diffusion of preservatives from topical dosage forms: a comparative study; Esposito E et al.; A study of the diffusion of parabens from topical formulations is presented here . In particular, four different topical formulations, namely, a water-in-oil emulsion, an oil-in-water emulsion, and two hydrophilic gels (Pemulen gel and Carbopol gel) were produced, containing a mixture of three common parabens, namely, methylparaben (MP), ethylparaben (EP), and propylparaben (PP) . An analytical method based on liquid extraction, followed by reversed-phase HPLC for the quantitative determination of MP, EP, and PP, was developed . The method allowed good separation of paraben mixtures and high percentages of recovery (> than 97%) . The diffusion kinetics of parabens from the produced formulations was determined by an in vitro system based on a Franz cell assembled with a synthetic membrane, followed by a reversed-phase HPLC analytical method . The comparative study demonstrated that, in the case of emulsions, diffusion coefficients are a function of the substituent of preservatives: the higher the solubility, the higher the diffusion of parabens . On the contrary, in the case of the hydrophilic gels, the higher the parabens solubility, the lower the diffusion coefficients . The method described here could represent a means of controlling the extent of diffusion of parabens from topical formulations in order to minimize percutaneous absorption and to control the availability of microbes.

Clin Microbiol Rev, 2003 Jul, 16(3), 517 - 33
Production of eicosanoids and other oxylipins by pathogenic eukaryotic microbes; Noverr MC et al.; Oxylipins are oxygenated metabolites of fatty acids . Eicosanoids are a subset of oxylipins and include the prostaglandins and leukotrienes, which are potent regulators of host immune responses . Host cells are one source of eicosanoids and oxylipins during infection; however, another potential source of eicosanoids is the pathogen itself . A broad range of pathogenic fungi, protozoa, and helminths produce eicosanoids and other oxylipins by novel synthesis pathways . Why do these organisms produce oxylipins? Accumulating data suggest that phase change and differentiation in these organisms are controlled by oxylipins, including prostaglandins and lipoxygenase products . The precise role of pathogen-derived eicosanoids in pathogenesis remains to be determined, but the potential link between pathogen eicosanoids and the development of TH2 responses in the host is intriguing . Mammalian prostaglandins and leukotrienes have been studied extensively, and these molecules can modulate Th1 versus Th2 immune responses, chemokine production, phagocytosis, lymphocyte proliferation, and leukocyte chemotaxis . Thus, eicosanoids and oxylipins (host or microbe) may be mediators of a direct host-pathogen "cross-talk" that promotes chronic infection and hypersensitivity disease, common features of infection by eukaryotic pathogens.

Ann Transplant, 2002, 7(4), 25 - 7
Differentiation of cellular reaction to alloantigens and bacterial infection in human skin graft--immunosuppressive drugs or antibiotics; Moscicka-Wesolowska M et al.; The human hand transplantations prompted revival of interest in evaluation of the rejection process of the grafted skin and its control with the antirejection drugs {1-3} . In case of first hand transplantation a combined immunosuppressive regimen was applied with currently available drugs resulting in acceptance of the entire composite graft . No major untoward systemic effects of antirejection therapy were observed . The most important clinical conclusion was that allogeneic skin can be accepted and function as in a normal extremity, although the attack of host cells on the graft can not be totally eliminated . Chronic perivascular and subepidermal infiltrates with recipient cells could be seen {4} . Another problem connected with skin transplantation is graft infection . Skin is inhabited by a specific spectrum of bacteria {5} . Allografted skin is more sensitive to bacterial penetration than normal skin due to local damage by the host-versus-graft cellular reaction and compromised immune reactivity to bacterial antigens by the immunosuppressive therapy . The histological pictures of rejecting skin represent a mixture of cellular reaction against the graft and penetrating microbes . Alloreaction requires modification of immunosuppressive regimen and infection is an indication for prolonged antibiotic therapy against skin bacterial flora . The question arises how to discriminate the alloreactive and bacterial changes in the skin graft . We studied the histological pictures of rejecting and infected human skin after transplantion to scid mice.

Health Phys, 2003 Jul, 85(1), 85 - 93
Developing a scientific basis for radiation risk estimates: goal of the DOE Low Dose Research Program; Brooks AL; The U.S . Department of Energy's Low Dose Radiation Research Program is a 10 y activity currently funded at $21 million per year . It focuses on biological responses to low doses (<0.1 Gy) of low-LET ionizing radiation . The overall goal of this program is to provide a sound scientific basis for the radiation protection standards . The program supports basic research that combines modern genomic, molecular, and cellular techniques with recent advances in scientific instrumentation . These combinations make it possible to detect responses and test paradigms associated with the mechanisms of low dose radiation action not previously measurable or testable . Research to date is briefly reviewed and suggests the need for some major paradigm shifts . Exposure of the extracellular matrix can modify both the pattern of gene expression and the phenotype of the cells which result in cell transformation without direct mutation . Low dose radiation exposure results in a range of dose-response relationships for changes in the number, types, and patterns of gene expression . Such studies suggest an increased role for gene expression relative to single mutations for radiation induced cancer . Low dose research using microbeams demonstrated that cells do not require a direct "hit" to result in significant biological alterations . These "bystander effects" demonstrate that "non-hit" cells respond with changes in gene expression, DNA repair, chromosome aberrations, mutations, and cell killing . Research to link genomic instability with cancer is also being conducted and will be discussed . Detection of radiosensitivity genes as markers of genetic susceptibility in individuals and populations can be used in epidemiological studies to determine how molecular changes may impact risk . It is not possible to determine how this research will influence current radiation standards . However, the Low Dose Research Program will help ensure that radiation standards are set using the best scientific data available, and that they are adequate and appropriate for the protection of workers and the public.

Health Phys, 2003 Jul, 85(1), 31 - 5
The bystander effect; Hall EJ; The bystander effect refers to the induction of biological effects in cells that are not directly traversed by a charged particle . The data available concerning the bystander effect fall into two quite separate categories, and it is not certain that the two groups of experiments are addressing the same phenomenon . First, there are experiments involving the transfer of medium from irradiated cells, which results in a biological effect in unirradiated cells . Second, there is the use of sophisticated single particle microbeams, which allow specific cells to be irradiated and biological effects studied in their neighbors; in this case communication is by gap junction . Medium transfer experiments have shown a bystander effect for cell lethality, chromosomal aberrations and cell cycle delay . The type of cell, epithelial vs . fibroblast, appears to be important . Experiments suggest that the effect is due to a molecule secreted by irradiated cells, which is capable of transferring damage to distant cells . Use of a single microbeam has allowed the demonstration of a bystander effect for chromosomal aberrations, cell lethality, mutation, and oncogenic transformation . When cells are in close contact, allowing gap junction communication, the bystander effect is a much larger magnitude than the phenomenon demonstrated in medium transfer experiments . A bystander effect has been demonstrated for both high- and low-LET radiations but it is usually larger for densely ionizing radiation such as alpha particles . Experiments have not yet been devised to demonstrate a comparable bystander effect on a three-dimensional normal tissue . Bystander studies imply that the target for the biological effects of radiation is larger than the cell and this could make a simple linear extrapolation of radiation risks from high to low doses of questionable validity.

Autoimmun Rev, 2003 May, 2(3), 115 - 8
The control of T cell activation vs . tolerance; Abbas AK; B7 costimulators and the T cell growth factor IL-2 are important stimuli for the activation of T lymphocytes and the development of effective immune responses . Recent studies show that the same signals promote the development of regulatory T cells or the apoptotic death of activated T cells, and thus, function to terminate immune responses and maintain self-tolerance . The balance between the different outcomes of the same signals may be determined by the magnitude of these signals and the presence or absence of concomitant stimuli, such as those generated during innate immune responses to microbes.

Immunol Cell Biol, 2003 Aug, 81(4), 289 - 96
Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells; Hodge S et al.; Chronic obstructive pulmonary disease is a highly prevalent, complex disease, usually caused by cigarette smoke . It causes serious morbidity and mortality and costs the global community billions of dollars per year . While chronic inflammation, extracellular matrix destruction and increased airway epithelial cell apoptosis are reported in chronic obstructive pulmonary disease, the understanding of the basic pathogenesis of the disease is limited and there are no effective treatments . We hypothesized that the accumulation of apoptotic airway epithelial cells chronic obstructive pulmonary disease in could be due to defective phagocytic clearance by alveolar macrophages . There have been no previous studies of the phagocytic capacity of alveolar macrophages in chronic obstructive pulmonary disease using physiologically relevant apoptotic airway epithelial cells as phagocytic targets . We developed a phagocytosis assay whereby cultured 16HBE airway epithelial cells were induced to apoptosis with ultraviolet radiation and stained with mitotracker green . Alveolar macrophages from bronchoalveolar lavage from eight control and six chronic obstructive pulmonary disease subjects were analysed following 1.5 h incubation with apoptotic airway epithelial cells, then staining with macrophage marker anti CD33 . CD33+/mitotracker green + events (i.e., alveolar macrophages which had phagocytosed apoptotic airway epithelial cells) were analysed using flow cytometry . Phagocytosis of polystyrene microbeads was investigated in parallel . A significantly reduced proportion of alveolar macrophages from chronic obstructive pulmonary disease subjects ingested apoptotic airway epithelial cells compared with controls (11.6 +/- 4.1% for chronic obstructive pulmonary disease versus 25.6 +/- 9.2% for control group) . Importantly, the deficiency was not observed using polystyrene beads, suggesting that the failure to resolve epithelial damage in chronic obstructive pulmonary disease may result, at least partially, from specific defects in phagocytic ability of alveolar macrophages to ingest apoptotic airway epithelial cells.

J Exp Med, 2003 Jul 7, 198(1), 133 - 44
Influenza virus-induced dendritic cell maturation is associated with the induction of strong T cell immunity to a coadministered, normally nonimmunogenic protein; Brimnes MK et al.; We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens . Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus . In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed . With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells . In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-gamma, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-gamma . The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus . CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules . The relatively slow (2-3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies . Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently.

Pest Manag Sci, 2003 Jun-Jul, 59(6-7), 671 - 80
Biological control of weeds: research by the United States Department of Agriculture-Agricultural Research Service: selected case studies; Quimby PC Jr et al.; Research by the USDA-Agricultural Research Service (ARS) on biological control of weeds has been practiced for many years because of its inherent ecological and economic advantages . Today, it is further driven by ARS adherence to Presidential Executive Order 13112 (3 February 1999) on invasive species and to USDA-ARS policy toward developing technology in support of sustainable agriculture with reduced dependence on non-renewable petrochemical resources . This paper reports examples or case studies selected to demonstrate the traditional or classical approach for biological control programs using Old World arthropods against Tamarix spp, Melaleuca quinquenervia (Cav) ST Blake and Galium spurium L/G aparine L, and the augmentative approach with a native plant pathogen against Pueraria lobata Ohwi = P montana . The examples illustrated various conflicts of interest with endangered species and ecological complexities of arthropods with associated microbes such as nematodes.

Nat Immunol, 2003 Aug, 4(8), 787 - 93 Epub 2003 Jul 06.
Crystal structure of peptidoglycan recognition protein LB from Drosophila melanogaster; Kim MS et al.; The family of peptidoglycan recognition proteins (PGRPs) are associated with the recognition of the peptidoglycan of microbes and subsequent activation of signaling pathways for immune response . Here the crystal structure of Drosophila PGRP-LB is determined at a resolution of 2.0 A and shows an active-site cleft with a zinc cage . Poor conservation of surface residues at the cleft predicts a widely varying individual specificity of PGRPs for molecular patterns on microbial cell walls . At the back of this cleft is a putatively conserved distinctive groove . The location and mainly hydrophobic nature of the groove indicate that the back face serves for subsequent signaling after clustering of PGRP molecules by binding to polymeric cell wall components.

J Microbiol Methods, 2003 Sep, 54(3), 423 - 6
GenomeComp: a visualization tool for microbial genome comparison; Yang J et al.; We have developed a software tool, GenomeComp, for summarizing, parsing and visualizing the genome sequences comparison results derived from voluminous BLAST textual output . With GenomeComp, the variation between genomes can be easily highlighted, such as repeat regions, insertions, deletions and rearrangements of genomic segments . This software provides a new visualizing tool for microbe comparative genomics.

Curr Biol, 2003 Jul 1, 13(13), 1122 - 8
Wound healing in the PU.1 null mouse--tissue repair is not dependent on inflammatory cells; Martin P et al.; Damage to neonatal and adult tissues always incites an influx of inflammatory neutrophils and macrophages . Besides clearing the wound of invading microbes, these cells are believed to be crucial coordinators of the repair process, acting both as professional phagocytes to clear wound debris and as a major source of wound growth factor signals . Here we report wound healing studies in the PU.1 null mouse, which is genetically incapable of raising the standard inflammatory response because it lacks macrophages and functioning neutrophils . Contrary to dogma, we show that these "macrophageless" mice are able to repair skin wounds with similar time course to wild-type siblings, and that repair appears scar-free as in the embryo, which also heals wounds without raising an inflammatory response . The growth factor and cytokine profile at the wound site is changed, cell death is reduced, and dying cells are instead engulfed by stand-in phagocytic fibroblasts . We also show that hyperinnervation of the wound site, previously believed to be a consequence of inflammation, is present in the PU.1 null wound, too.

Ann Allergy Asthma Immunol, 2003 Jun, 90(6 Suppl 3), 59 - 63
Breast-feeding, infant formulas, and the immune system; Hanson LA et al.; OBJECTIVE: Breast-feeding provides many advantages to the offspring, but presently there is an ongoing debate whether or not it prevents allergy any better than certain formulas . This report reviews the mechanisms involved and discusses how breast-feeding may protect against allergy . DATA SOURCES: The review builds on an internet-based literature search in addition to our own data . RESULTS: Human milk is the food best adapted to the needs of the offspring, also because it provides efficient protection against infections and actively stimulates the development of the infant's own immune system . The major host defense system is provided via the secretory IgA antibodies produced in the mammary glands by lymphocytes, which have migrated there from the mother's gut mucosa . Therefore, these antibodies in the milk are primarily directed against the microbes in the mother's gut and her food proteins . As a result, breast-feeding starting directly after delivery will provide an excellent defense against the microbes normally meeting the neonate and needed to induce development of its immune system . The milk also contains numerous components, which seem to enhance the infant's host defense as well as capacity to develop tolerance, helping to avoiding allergic reactivity to foods, etc . CONCLUSIONS: Several studies show that breast-feeding prevents allergic diseases, but there are also good disagreeing studies . Supported by animal data, it seems that protection is enhanced in areas with more advantageous fat intake, inter alia lower ratio of n-6/n-3 fatty acids . Breast-feeding seems to protect against future development of allergic diseases, but possibly less so in countries with an untoward maternal fat intake.

Trends Cardiovasc Med, 2003 Jul, 13(5), 201 - 6
Endothelial progenitor cells: isolation and characterization; Hristov M et al.; Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs) . Of the three cell markers (CD133, CD34, and the vascular endothelial growth factor receptor 2) that characterize the early functional EPCs, located predominantly in the bone marrow, EPCs obviously lose CD133/CD34 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor when migrating to the circulation . Various isolation procedures of EPCs from different sources by using adherence culture or magnetic microbeads have been described, but published findings with regard to the number of EPCs in the peripheral circulation of healthy adults are scanty and no data regarding the lifetime of EPCs in vivo exist . Clinical studies employing EPCs for neovascularization of ischemic organs have just been started; however, the mechanisms stimulating or inhibiting the differentiation of bone marrow-derived EPCs in vivo and the signals causing their adhesion, migration, and homing to sites of injured tissue are largely unknown at present.

Opt Lett, 2003 Jun 15, 28(12), 998 - 1000
Periodic microbending-induced core-to-cladding mode coupling in polarization-maintaining fibers; Lin CH et al.; We investigate codirectional mode coupling induced by periodic microbending in polarization-maintaining (PM) fibers by using both an acoustic flexural wave and a pair of corrugated fixtures . The measured filtering spectra are found to be strongly polarization dependent . In addition, the coupling strength depends on the angle between the birefringence axes and the bending direction . These characteristics can be utilized for a variety of applications, such as fiber polarizers, polarization-dependent-loss compensators, and on-line alignment of birefringence axes for the manufacture of PM fiber couplers.

Ying Yong Sheng Tai Xue Bao, 2003 Mar, 14(3), 349 - 53
{Effect of ecological organic fertilizer on tomato bacterial wilt and soil microbial diversities}; Cai Y et al.; The effects of ecological organic fertilizer (EOF) on tomato bacterial wilt and soil microbial community were studied through field trial and fatty acid methyl esters (FAMEs) analysis . The results showed that the incidence rate of tomato bacterial wilt was 100% in continuous cropped soil, and significantly reduced to 39% and 50% when applying EOF at 1.0 kg.m-2 and 0.5 kg.m-2, respectively . There was no significant difference between two fertilization treatments . The fatty acid fingerprints of soil microbial community were greatly changed, and the relative contents of cyc17:0 cited as biomarker for GN bacteria, c18:2 w6 as biomarker for fungi and c16:1 11c as biomarker for arbuscular mycorrhizal (AM) fungi all increased when applying EOF . The minimum distance of cluster analysis was 1.08 between control and treatments of applying ecological organic fertilizer, and 0.70 between different fertilization treatments . Applying EOF could adjust soil microbial community structure, benefit for soil beneficial microbes, and develop stability and disease suppression of soil ecological system, and hence, improve soil quality.

Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8454 - 9 Epub 2003 Jun 27.
Does a betaretrovirus infection trigger primary biliary cirrhosis?
Xu L, Shen Z, Guo L, Fodera B, Keogh A, Joplin R, O'Donnell B, Aitken J, Carman W, Neuberger J, Mason A.
Patients with primary biliary cirrhosis develop progressive ductopenia associated with the production of antimitochondrial antibodies that react with a protein aberrantly expressed on biliary epithelial cells and peri-hepatic lymph nodes . Although no specific microbe has been identified, it is thought that an infectious agent triggers this autoimmune liver disease in genetically predisposed individuals . Previous serologic studies have provided evidence to suggest a viral association with primary biliary cirrhosis . Here we describe the identification of viral particles in biliary epithelium by electron microscopy and the cloning of exogenous retroviral nucleotide sequences from patients with primary biliary cirrhosis . The putative agent is referred to as the human betaretrovirus because it shares close homology with the murine mammary tumor virus and a human retrovirus cloned from breast cancer tissue . In vivo, we have found that the majority of patients with primary biliary cirrhosis have both RT-PCR and immunohistochemistry evidence of human betaretrovirus infection in lymph nodes . Moreover, the viral proteins colocalize to cells demonstrating aberrant autoantigen expression . In vitro, we have found that lymph node homogenates from patients with primary biliary cirrhosis can induce autoantigen expression in normal biliary epithelial cells in coculture . Normal biliary epithelial cells also develop the phenotypic manifestation of primary biliary cirrhosis when cocultivated in serial passage with supernatants containing the human betaretrovirus or the murine mammary tumor virus, providing a model to test Koch's postulates in vitro.

Curr Opin Microbiol, 2003 Jun, 6(3), 274 - 81
Cultivation of recalcitrant microbes: cells are alive, well and revealing their secrets in the 21st century laboratory; Leadbetter JR; Any talk of the demise of in vitro cultivation as a useful mechanism for revealing many of nature's past and present secrets appears to be unfounded and premature . The first years of this century have been as productive in the cultivation of physiologically novel, environmentally abundant and phylogenetically distinct microbes as were the first years of the 20th century . The diversity of organic and inorganic electron donors and acceptors known to be used during microbial energy metabolisms continues to grow, expanding our appreciation for the niches that may be, or historically may have been, filled by microbes in the biosphere . Either guided and instigated by, or independent of, the results of gene inventories representing diverse environmental settings, significant advances are constantly being made in the isolation of bacteria and archaea, demonstrating either strikingly rich phylogenetic diversity or significant activity and abundance in their respective environments . The potential synergisms between molecular ecological analyses and innovative in vitro growth studies are real and should be embraced, rather than treated as dueling agents in some zero-sum game.

J Biomech, 2003 Aug, 36(8), 1135 - 41
Regional epicardial strain in the embryonic chick heart during the early looping stages; Alford PW et al.; Epicardial strains were measured in Hamburger-Hamilton stage 11 and 12 embryonic chick hearts (1.6-2.0 days of incubation) . These stages include part of the early phase of cardiac looping, as the initially straight heart tube bends and twists to form a curved c-shaped tube . By analyzing the motion of microbeads placed on the myocardial surface, we measured strains near the outer curvature, in the central region, and near the inner curvature of the primitive ventricle . No significant differences in strain were found between stages . Relative to end diastole, all three regions shortened by about 10% during systole in the circumferential direction, and the outer curvature shortened longitudinally by about 5% . In contrast, and unlike strains in older hearts, the inner curvature and central regions elongated by approximately 5-10% in the longitudinal direction during systole . These results are consistent with microstructural data and suggest that the material properties of the outer curvature are relatively isotropic, whereas the properties of the central and inner curvature regions are orthotropic, with contractile stress exerted primarily in the circumferential direction.

J Endotoxin Res, 2003, 9(3), 176 - 80
Macrophage recognition of zymosan particles; Underhill DM; Zymosan particles have served as a model for recognition of microbes by the innate immune system for over 50 years . Zymosan induces inflammatory signals in macrophages through Toll-like receptors TLR2 and TLR6 . In addition, phagocytic receptors on macrophages bind zymosan and stimulate particle engulfment . We have further examined the requirements for induction of inflammatory responses such as TNF-alpha production and NF-kappaB activation by zymosan in mouse macrophages . We have observed that direct particle contact is required (excluding a role for soluble components of zymosan preparations) and that contact with a single particle is sufficient to trigger cytokine production . Further, ablation of the Toll-like receptor-stimulating activity of zymosan does not affect the ability of phagocytic receptors to internalise the particle.

J Endotoxin Res, 2003, 9(3), 169 - 75
TLR2 and TLR4 agonists stimulate unique repertoires of host resistance genes in murine macrophages: interferon-beta-dependent signaling in TLR4-mediated responses; Toshchakov V et al.; That TLRs share a common MyD88-dependent signaling pathway which results in the generation of nuclear DNA-binding proteins, such as NF-kappaB, is a well-accepted paradigm . However, studies from our laboratories and others suggested that TLR4 agonists elicit a more diverse pattern of gene expression in murine macrophages than TLR2 agonists . The data presented show that activation of TLR4 by Escherichia coli LPS results in an MyD88-independent, TIRAP/Mal-dependent signaling pathway that, in turn, leads to early induction of interferon-beta (IFN-beta) . IFN-beta, in turn, acts in an autocrine/paracrine fashion on the macrophage to activate STAT1-containing DNA binding complexes that participate in the induction of genes not expressed in response to natural or synthetic TLR2 agonists . These data support the hypothesis that the host response to microbes is controlled by TLRs at two levels: (i) the "sensing" of differences in microbial structures through the TLR extracellular domain; and (ii) signaling pathways that are initiated via interactions through unique intracytoplasmic regions of different TLRs with adaptor proteins.

Environ Sci Technol, 2003 Jun 1, 37(11), 2371 - 5
Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza; Joner EJ et al.; Phytoremediation of organic pollutants depends on plant-microbe interactions in the rhizosphere, but the extent and intensity of such rhizosphere effects are likely to decrease with increasing distance from the root surface . We conducted a time-course pot experiment to measure dissipation of polycyclic aromatic hydrocarbons (PAHs) in the rhizosphere of clover and ryegrass grown together on two industrially polluted soils (containing 0.4 and 2 g kg(-1) of 12 PAHs) . The impact of the fungal root symbiosis arbuscular mycorrhiza (AM) on PAH degradation was also assessed, as these fungi have previously improved plant establishment on PAH-polluted soils and enhanced PAH degradation in spiked soil . The two soils behaved differently with respect to the time-course of PAH dissipation . The less polluted and more highly organic soil showed low initial PAH dissipation rates, with small positive effects of plants after 13 weeks . At the final harvest (26 weeks), the amounts of PAHs extracted from nonplanted pots were higher than the initial concentrations . In parallel planted pots, PAH concentrations decreased as a function of proximity to roots . The most polluted soil showed higher initial PAH dissipation (25% during 13 weeks), but at the final harvest PAH concentrations had increased to values between the initial concentration and those at 13 weeks . An effect of root proximity was observed for the last harvest only . The presence of mycorrhiza generally enhanced plant growth and favored growth of clover at the expense of ryegrass . Mycorrhiza enhanced PAH dissipation when plant effects were observed.

Am J Respir Crit Care Med, 2003 Sep 15, 168(6), 685 - 91 Epub 2003 Jun 26.
Bronchoalveolar lavage fluid surfactant protein-A and surfactant protein-D are inversely related to inflammation in early cystic fibrosis; Noah TL et al.; The pulmonary collectins surfactant protein (SP)-A and SP-D play important roles in innate lung defense, enhancing opsonization of microbes and limiting lung inflammatory responses . To quantify relationships among collectins, bacteria, and inflammation in early cystic fibrosis (CF) airway secretions, bronchoalveolar lavage fluids (BALFs) were collected from children undergoing clinically indicated bronchoscopy . Quantitative bacteriology, differential cell counts, and ELISA for SP-A and SP-D were assessed . Significantly increased numbers of neutrophils relative to bacteria were noted in BALF from CF compared with non-CF subjects . Although SP-A levels tended to be lower in CF compared with non-CF, this was only significant in the presence of bacterial infection . Among CF patients, SP-A concentrations in BALF were inversely related to inflammation, bacterial colony-forming units per milliliter, and age . SP-D levels were significantly decreased in CF patients, and SP-D was rarely detectable in the presence of infection . Among CF patients, SP-D correlated inversely with inflammation and bacterial colony-forming units per milliliter, and there was decreased immunostaining of BALF cells for SP-D in CF . Immunohistochemistry of CF autopsy lung sections for SP-A and SP-D confirmed their paucity at sites of infection and inflammation . We conclude that relative collectin deficiency occurs early in CF airways and is inversely related to inflammation in CF airways.

FEMS Microbiol Rev, 2003 Jun, 27(2-3), 427 - 47
Microbial ferric iron reductases; Schroder I et al.; Almost all organisms require iron for enzymes involved in essential cellular reactions . Aerobic microbes living at neutral or alkaline pH encounter poor iron availability due to the insolubility of ferric iron . Assimilatory ferric reductases are essential components of the iron assimilatory pathway that generate the more soluble ferrous iron, which is then incorporated into cellular proteins . Dissimilatory ferric reductases are essential terminal reductases of the iron respiratory pathway in iron-reducing bacteria . While our understanding of dissimilatory ferric reductases is still limited, it is clear that these enzymes are distinct from the assimilatory-type ferric reductases . Research over the last 10 years has revealed that most bacterial assimilatory ferric reductases are flavin reductases, which can serve several physiological roles . This article reviews the physiological function and structure of assimilatory and dissimilatory ferric reductases present in the Bacteria, Archaea and Yeast . Ferric reductases do not form a single family, but appear to be distinct enzymes suggesting that several independent strategies for iron reduction may have evolved.

FEMS Microbiol Rev, 2003 Jun, 27(2-3), 239 - 61
Nickel uptake and utilization by microorganisms; Mulrooney SB et al.; Nickel is an essential nutrient for selected microorganisms where it participates in a variety of cellular processes . Many microbes are capable of sensing cellular nickel ion concentrations and taking up this nutrient via nickel-specific permeases or ATP-binding cassette-type transport systems . The metal ion is specifically incorporated into nickel-dependent enzymes, often via complex assembly processes requiring accessory proteins and additional non-protein components, in some cases accompanied by nucleotide triphosphate hydrolysis . To date, nine nickel-containing enzymes are known: urease, NiFe-hydrogenase, carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase, methyl coenzyme M reductase, certain superoxide dismutases, some glyoxylases, aci-reductone dioxygenase, and methylenediurease . Seven of these enzymes have been structurally characterized, revealing distinct metallocenter environments in each case.

Best Pract Res Clin Gastroenterol, 2003 Aug, 17(4), 679 - 94
Alcohol consumption and cancer of the gastrointestinal tract; Salaspuro MP; Excessive alcohol consumption and heavy smoking are the main risk factors for upper digestive tract cancers . Cancer risk is dose-dependent and alcohol and smoking have synergistic effects . Alcohol is not carcinogenic . However, its first metabolite-acetaldehyde-has recently been shown to be a local carcinogen in humans . Microbes representing normal human gut flora are able to produce acetaldehyde from ethanol . This results in high local acetaldehyde concentrations in the saliva and contents of the large intestine . Asian heavy drinkers with a genetic deficiency for detoxifying acetaldehyde form an exceptional human 'knockout' model for long-term acetaldehyde exposure . The risk of alcohol-related digestive tract cancers is particularly high among this population . All mechanisms that have an effect on salivary or intracolonic acetaldehyde concentration are of importance . The message for prevention is that one should take care to have good oral hygiene and to avoid smoking, heavy drinking and drinking to intoxication.

Best Pract Res Clin Gastroenterol, 2003 Aug, 17(4), 543 - 55
Alcohol-related diseases of the mouth and throat; Riedel F et al.; Chronic consumption of alcoholic beverages is an accepted social custom worldwide . In the upper aerodigestive tract, local morphological, metabolic and functional alterations can be present as a result of alcohol consumption . A clinical link between the chronic consumption of alcohol and head and neck cancer has been observed for decades . While alcohol was described initially as a risk enhancer only in smokers, a number of epidemiological studies have now provided sufficient evidence that chronic alcohol consumption increases the risk of head and neck cancer independent of exposure to tobacco smoke . Systemic effects of alcohol interact with local changes in the morphology and function of the salivary glands . In addition, alcohol leads to the accumulation of pathological microbes within the mucosa, leading to chronic infection . Susceptibility to carcinogens and cell proliferation in the mucosa are increased, resulting in genetic changes with the development of dysplasia, leucoplakia and carcinoma . Chronic alcohol consumption has been correlated with an increased risk of cancer and increased mortality in a dose-effect relationship . A number of biologically plausible mechanisms exist by which alcohol may cause cancer . These mechanisms are discussed in this chapter.

CNS Drugs, 2003, 17(9), 669 - 75; discussion 676-7
SLI-381 (Adderall XR); McKeage K et al.; SLI-381 is an extended-release formulation of short-acting Adderall, a racemic mixture of dextro- and levo-isomers of amphetamine salts . Drug-containing microbeads within the SLI-381 capsule give a double-pulsed delivery, similar to that achieved by two equal doses of the short-acting formulation administered 4 hours apart . In an intent-to-treat analysis of a 3-week, double-blind study in 563 children with attention-deficit hyperactivity disorder (ADHD), SLI-381 10, 20 or 30 mg once daily improved mean morning and afternoon behaviour scores compared with baseline significantly more than placebo (p < 0.001 for all comparisons), as assessed by the Connors Global Index Scale for teachers (CGIS-T) . Following treatment, CGIS-T scores were similar to those reported in children without ADHD . In the same study, a dose-response relationship was observed, and increasing the dosage of SLI-381 by 10mg at weekly intervals, to a maximum of 30 mg once daily, resulted in further improvements in the scores of the CGIS-T . After early morning administration of SLI-381 in this double-blind study, late-afternoon scores of the CGIS for parents were similar to morning scores . SLI-381 was generally well tolerated in randomised trials in children with ADHD for up to 24 months . Overall, adverse events were mild to moderate in intensity.

Cytometry B Clin Cytom, 2003 Jul, 54(1), 54 - 7
Immune consequences of pediatric and adult cardiovascular surgery: report of the 7th Leipzig workshop; Tarnok A et al.; Cardiovascular surgery in children and adults is among the most common types of interventions in the western hemisphere for innate and acquired defects . In the recent decades, the risk of cardiovascular surgery has been reduced substantially . Nevertheless, open heart surgery is risky for the patient and can lead to postoperative complications such as postpericardiotomy syndrome, capillary leak syndrome, or multiple organ failure . To gain further understanding into the response to cardiovascular surgery, it is necessary to join forces from several disciplines of medicine and natural sciences . Interdisciplinarity is the basic concept of the Leipzig Workshop . The consensus of the workshop was that cardiovascular surgery with cardiopulmonary bypass induces a systemic antiinflammatory response due to (a) elimination of activated cells, (b) compensatory reaction to a local proinflammatory responses, (c) interleukin-10 release, (d) anesthetics and medication, and (e) leukocyte extravasation . The subsequent proinflammatory reaction is the response to surgical trauma modulating the antiinflammatory reaction . Novel therapeutic approaches include the introduction of autologous endothelial progenitor cells from the peripheral blood into the sites of injury . The analysis of immune response and outcome prediction require novel analytical tools that allow fast, accurate, and quantitative determination of the desired parameters in a multiplexed manner (i.e., cytomics), such as flow cytometric microbead array assays and slide-based cytometry . The major goal is predictive medicine by cytomics, i.e., the individualized risk assessment by analyzing the cytome in combination with sophisticated data pattern recognition . These developments may lead to individualized therapy for the benefit of the patient and cost reduction .

Cell Tissue Res, 2003 Jul, 313(1), 47 - 53 Epub 2003 Jun 24.
Distribution of two types of lymphocytes (intraepithelial and lamina-propria-associated) in the murine small intestine; Tamura A et al.; The intestine, which is exposed to nutrition and to food-derived antigens and microbes including viruses and bacteria, might be an important site for the immune response . Crucial structural and functional differences exist between the small and large intestine, regional differences even having been demonstrated within the small intestine . Accordingly, intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) might be heterogeneous among the different intestinal regions . The aim of this study has been to describe, as accurately as possible, the numbers and T-cell receptor (TCR) phenotypes of IELs and LPLs present in distinct regions of the murine small intestine under physiological conditions . Using an immunohistological technique to differentiate IELs from LPLs, the differential enumeration of IELs and LPLs in distinct regions of the murine small intestine, based upon their definition originally determined by their location, has been performed for the first time and has demonstrated that (1) there are more IELs than LPLs in the duodenum and jejunum, but more LPLs than IELs in the ileum, (2) in the duodenum and jejunum, TCRgammadelta IELs account for 70%-75% of the total CD3(+) IELs, a much greater percentage than previously reported, (3) the ratio of TCRgammadelta to TCRalphabeta IELs is inverted in the ileum, with more than 75% IELs being TCRalphabeta-positive, (4) the lamina propria forms one functional unit throughout the small intestine in terms of the TCR subset components (TCRalphabeta:TCRgammadelta=3:1), and (5) the ileum is entirely different from other regions of the small intestine . To deepen our understanding of the functional significance of the small intestine as an immunologically competent organ, the precise distributions of IELs and LPLs, the ratio of their various subsets, and the strict distinction of IELs and LPLs, as described in this study, is indispensable.

Nucleic Acids Res, 2003 Jul 1, 31(13), 3727 - 8
AHMII: Agent to Help Microbial Information Integration; Sugawara H et al.; Reproducibility is the key to science . Therefore, the fidelity of organisms, particularly type strains, used in molecular biology must be authenticated before embarking on a series of experiments . In the case of microbes the authentic strain can be obtained from a culture collection . However, tracking down a culture collection that possesses the strain can be an arduous task . With this in mind we have developed a one-stop search engine for bacteria, fungi, yeasts and cell lines that indicates which collections carry the strain of interest . The search agent is named Agent to Help Microbial Information Integration (AHMII) and the URL address is http://www.wdcm.org/AHMII/ahmii.html.

Med Arh, 2003, 57(2), 71 - 4
Q-fever serologic diagnostics with inhabitants of Canton of Sarajevo 2001 year; Beslagic E et al.; Q-fever is widely spread antropoozosis caused by Coxiellae burnetti, an intracellular compulsory microbe parasite . Two characteristics of Coxiellae burnetti are of crucial importance for appearance of Q-fever, especially in the circumstances when the cause of infection remains unclear . The first one is a high resistance of Coxiellae burnetti to environment changes and the second one is a small size of the infectious dose . The clinical manifestations of Q-fever can vary, so the making of diagnosis is still based on serology, with Phase I and Phase II antibodies and the difference between the acute and the chronic form of the disease . Serologic diagnostics presents the method of choice with Q-fever (IFT) in serums of patients with clinical suspicion to Coxiellae burnetti infection . We have tested the serums of patients from Canton of Sarajevo which were coming to our laboratory from January 2001-December 2001 . Out of 58 processed serums the specific IgM antibodies were found in 10 serums and specific IgG antibodies in 27 serums.

Cytometry A, 2003 Jul, 54(1), 27 - 35
Rapid detection of respiring Escherichia coli O157:H7 in apple juice, milk, and ground beef by flow cytometry; Yamaguchi N et al.; BACKGROUND: Rapid and simple methods to detect viable pathogenic microbes in foods and drinks are required . Flow cytometry was used for the rapid detection of respiring Escherichia coli O157:H7 cells in apple juice, milk, and ground beef . METHODS: CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was used to estimate the respiratory activity of bacteria . Fluorescein isothiocyanate (FITC)-labeled anti-E . coli O157:H7 direct antibody (FA) was used for the specific detection of target cells . Food samples were inoculated with starved E . coli O157:H7 and E . coli K-12 cells, and analyzed by both fluorescent microscopy and flow cytometry after double staining with FA and CTC . RESULTS: Respiring E . coli O157:H7 cells in food samples showed strong fluorescence of both FA (green) and CTC (red); thus, they could be clearly and specifically distinguished from respiring E . coli K-12 or inactive cells . A good correlation was achieved in flow cytometric analysis between the numbers of inoculated viable E . coli O157:H7 and those detected in milk and apple juice . The detection threshold for this flow cytometry for E . coli O157:H7 in milk, apple juice, and ground beef was 10(3) cells/ml (milk and apple juice) or 10(3) cells/g (ground beef) of sample when the total bacterial number in the sample was 10(6) cells/ml . CONCLUSIONS: Respiring E . coli O157:H7 in food samples can be detected specifically within a few hours . Flow cytometry with FA-CTC double staining can be used to examine food contamination with various pathogenic microbes demonstrating physiologic activity through the use of a suitable fluorescent antibody .

Genome Res, 2003 Jun, 13(6B), 1376 - 88
Comparative analysis of apoptosis and inflammation genes of mice and humans; Reed JC et al.; Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology . Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFkappaB induction . Proteins involved in apoptosis and NFkappaB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods . Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFkappaB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins . After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families . Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs . This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes . With this caveat, we discuss similarities and differences in human and murine genes from these domain families.

Infect Immun, 2003 Jul, 71(7), 3885 - 93
Analysis of gene expression in ceca of Helicobacter hepaticus-infected A/JCr mice before and after development of typhlitis; Myles MH et al.; The inflammatory bowel diseases, Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract . The causes of these diseases remain unknown; however, prevailing theories suggest that chronic intestinal inflammation results from a dysregulated immune response to ubiquitous bacterial antigens . While a substantial body of data has been amassed describing the role of the adaptive immune system in perpetuating and sustaining inflammation, very little is known about the early signals, prior to the development of inflammation, that initiate and direct the abnormal immune response . To this end, we characterized the gene expression profile of A/JCr mice with Helicobacter hepaticus-induced typhlitis at month 1 of infection, prior to the onset of histologic disease, and month 3 of infection, after chronic inflammation is fully established . Analysis of the gene expression in ceca of H . hepaticus infected mice revealed 25 up-regulated and 3 down-regulated genes in the month-1 postinoculation group and 31 up-regulated and 2 down-regulated genes in the month-3 postinoculation group . Among these was a subset of immune-related genes, including interferon-inducible protein 10, monokine induced by gamma interferon, macrophage-induced protein 1 alpha, and serum amyloid A1 . Semiquantitative real-time reverse transcriptase PCR confirmed the increased expression levels of these genes, as well as elevated expression of gamma interferon . To our knowledge, this is the first report profiling cecal gene expression in H . hepaticus-infected A/JCr mice . The findings of altered gene expression prior to the development of any features of pathology and the ensuing chronic disease course make this an attractive model for studying early host response to microbe-induced inflammatory bowel disease.

Cryobiology, 2003 Jun, 46(3), 205 - 29
Protectants used in the cryopreservation of microorganisms; Hubalek Z; The cryoprotective additives (CPAs) used in the frozen storage of microorganisms (viruses, bacteria, fungi, algae, and protozoa) include a variety of simple and more complex chemical compounds, but only a few of them have been used widely and with satisfactory results: these include dimethylsulfoxide (Me2SO), glycerol, blood serum or serum albumin, skimmed milk, peptone, yeast extract, saccharose, glucose, methanol, polyvinylpyrrolidone (PVP), sorbitol, and malt extract . Pairwise comparisons of the cryoprotective activity of the more common CPAs used in cryomicrobiology, based on published experimental reports, indicate that the most successful CPAs have been Me2SO, methanol, ethylene glycol, propylene glycol, and serum or serum albumin, while glycerol, polyethylene glycol, PVP, and sucrose are less successful, and other sugars, dextran, hydroxyethyl starch, sorbitol, and milk are the least effective . However, diols (as well as some other CPAs) are toxic for many microbes . Me2SO might be regarded as the most universally useful CPA, although certain other CPAs can sometimes yield better recoveries with particular organisms . The best CPA, or combination of CPAs, and the optimum concentration for a particular cryosensitive microorganism has to be determined empirically . This review aims to provide a summary of the main experimental findings with a wide range of additives and organisms . A brief discussion of mechanisms of CPA action is also included.

J Immunol, 2003 Jul 1, 171(1), 69 - 77
Thy-1 signaling in the context of costimulation provided by dendritic cells provides signal 1 for T cell proliferation and cytotoxic effector molecule expression, but fails to trigger delivery of the lethal hit; Haeryfar SM et al.; Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis . However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses . In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells . Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb . This effect was not dependent on dendritic cell Fcgamma receptors, but was a result of B7-mediated costimulation (signal 2) . T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively . Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery . Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays . We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function . The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

Methods Cell Sci, 2002, 24(1-3), 79 - 90
Host-pathogen interactions: leukocyte phagocytosis and associated sequelae; Voyich JM et al.; Polymorphonuclear leukocytes (PMNs) are a critical component of the human innate immune response and are the first line of defense against invading microorganisms . Phagocytosis of invading microbes induces production of reactive oxygen species (ROS) by PMNs, which facilitates bactericidal activity . In addition to eliminating microorganisms, phagocytosis also accelerates PMN apoptosis, a process critical for resolution of inflammation . Inasmuch as leukocyte phagocytosis and ROS production are key components of the innate immune response, we developed flow cytometric methods to evaluate these processes in human PMNs . In contrast to traditional microscopy-based analyses, the methods described herein provide objective and high throughput measures of host cell-pathogen interactions . Importantly, they can be adapted for use with a number of fluorometric probes, and bacterium and host cell of choice, and each is based upon a common phagocytosis assay system . We also describe methods to measure phagocytosis-induced PMN apoptosis with this assay system . These methods entail detecting surface-exposed phosphatidylserine (early apoptosis), and measuring PMN chromatin condensation and DNA fragmentation (late apoptosis) . Taken together, these assays provide rapid and accurate assessment of critical PMN processes.

J Theor Biol, 2003 Jul 21, 223(2), 149 - 60
Optimal choice between feedforward and feedback control in gene expression to cope with unpredictable danger; Shudo E et al.; All organisms face risks of unpredictable danger caused by harmful physical environments, pathogens, parasites or predators . Organisms may have several alternative ways of coping with such dangers . These differ in cost, effectiveness and activation time . We study the conditions under which it is optimal to use different alternatives for damage control . As an example we consider a microbe (such as E . coli), which may experience heat shocks that cause denaturation of proteins in the cell . To restore the denatured proteins the organism produces heat-shock proteins (HSP) . There are two different pathways for production of HSP . Some HSP are produced immediately after a heat shock (feedforward control), but additional HSP may be produced thereafter, stimulated by the presence of denatured proteins (feedback control) . Feedforward is based solely on heat-shock intensity without accurate information on the resulting amount of denatured proteins . We examine the optimal combination of the two pathways that minimizes the sum of the damage caused by the presence of untreated denatured proteins and the production cost of HSP . The optimal response depends on the time delay for feedback control, the effectiveness of HSP in processing denatured proteins, the production cost of HSP, the severity of damage by denatured proteins and the probability distribution of the abundance of denatured protein conditional on heat-shock intensity . We find that feedforward control should always be used . Additional HSP may be produced by feedback control when the abundance of denatured protein is large whilst no feedback control should be used when it is small . All the HSP are produced by feedforward control when the maximum is close to the mean of denatured protein abundance conditional on the heat-shock intensity.

Biotechniques, 2003 Jun, 34(6), 1238 - 43
Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR; Meimberg H et al.; Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis . Using these methods, we were able to select single chloroplasts of Nicotiana tabacum . Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide . Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube . Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast . The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

Biochemistry, 2003 Jun 24, 42(24), 7497 - 508
The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli; Price JC et al.; The Fe(II)- and alpha-ketoglutarate(alphaKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both . A consensus mechanism for these enzymes, involving (i) addition of O(2) to a five-coordinate, (His)(2)(Asp)-facially coordinated Fe(II) center to which alphaKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O(2) on the ketone carbonyl of alphaKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O(2)(-)) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O(2)(-) complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O(2) has ever been detected . In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods . Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature gamma-radiolysis of the trapped intermediate) by Mossbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex . Its Mossbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation . The properties of the complex and previous results on related alphaKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O(2)-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O(2)(-) species . The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.

J Appl Microbiol, 2003, 95(1), 120 - 8
Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water; Ogawa M et al.; AIMS: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment . METHODS AND RESULTS: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed . This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens . An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed . This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r2 = 0.96 and 0.93, respectively) . CONCLUSION: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h . SIGNIFICANCE AND IMPACT OF THE STUDY: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time . This system could be easily applied to the rapid analysis of microbes in various environments.

J Appl Microbiol, 2003, 95(1), 109 - 19
Comparison of microbial communities in four different composting processes as evaluated by denaturing gradient gel electrophoresis analysis; Ishii K et al.; AIMS: We aimed to systematically understand the composting processes by a comparison of microbial communities during four full-scale composting processes . METHODS AND RESULTS: Microbial communities during the four different full-scale composting processes were analysed by denaturing gradient gel electrophoresis combined with measurement of physicochemical parameters . Two composting processes utilized sewage sludge and two utilized food-waste . Comparison of the four processes indicated that the concentration of dissolved organic carbon was higher in the food-waste-composting than in the sewage-sludge-composting processes, and microbial communities varied with composting substrate . The tendency for different microbes to appear in the composting process with different concentrations of dissolved organic carbon agreed with a previous study that showed that microbial succession occurred with a decrease in dissolved organic carbon in a laboratory-scale food-waste-composting process . CONCLUSIONS: Our results suggested that the main factor affecting microbial communities in the composting process is the concentration of dissolved organic materials . SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to studying microbial communities involved in composting, this research is also the first to study composting mechanisms using molecular methods . The results of our studies may be helpful in the design and management of composting processes.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7616 - 21 Epub 2003 Jun 12.
Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening; Lammerding J et al.; The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1 . To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells . For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time . In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening . Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation . Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

Ultramicroscopy, 2003 Oct-Nov, 97(1-4), 359 - 63
Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell; Kim H et al.; Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM) . First, the distribution of the integrin beta(5) subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell . To visualize the distribution of the receptor on a living cell by an independent and potentially a more quantitative method, the AFM was used with a microbead attached to the cantilever tip to increase the area of contact and VN was immobilized on the microbead . Force measurements were then performed over a large area of a living murine osteoblastic cell using the microbead covered with VN.

Gene, 2003 May 22, 310, 175 - 83
Characterization of thioredoxin reductase genes (trr1) from Pneumocystis carinii and Pneumocystis jiroveci; Kutty G et al.; We have characterized the thioredoxin reductase (trr1) genes from Pneumocystis carinii and Pneumocystis jiroveci, and have demonstrated that multiple copies of an approximately 500 base pair fragment of the trr1 gene are present in P . carinii, but not in P . jiroveci . Thioredoxin reductases encoded by the full-length genes have predicted molecular weights of approximately 35,000 and show high homology to yeast Trr1 . An NADPH-binding domain with a putative redox active site CAVC as well as an flavin-adenine dinucleotide-binding domain are highly conserved in both proteins, which were 85% identical . The multicopy trr1 gene fragments in P . carinii are not transcribed or expressed . Duplication of the gene fragment likely occurred in conjunction with duplication of the kexin homologue, protease-1, which is located immediately upstream of the trr1 gene . Thioredoxin reductase, an enzyme implicated in the growth, survival and pathogenicity of certain microbes, could be a potential target for therapeutic intervention in Pneumocystis infection.

Pediatr Crit Care Med, 2001 Apr, 2(2), 127 - 132
Frequency of change of ventilator circuit in premature infants: Impact on ventilator-associated pneumonia; Makhoul IR et al.; OBJECTIVE: Ventilator-associated pneumonia (VAP) is associated with substantial mortality . The frequency of changing the ventilator circuit (VC) might influence the occurrence rate of VAP . In premature infants receiving ventilatory support, the question regarding the frequency of changing VC is as yet unsettled . DESIGN: A prospective, randomized, and controlled trial in 60 premature neonates receiving ventilatory support . INTERVENTIONS: We investigated the impact of two VC change regimens on VAP in premature infants, either every 24 hrs or every 72 hrs . In each patient, the humidifier, inspiratory tube, and expiratory tube were changed and cultured at the assigned intervals along with cultures of tracheal aspirates . Blood cultures were obtained whenever there was clinical evidence of pneumonia or sepsis . MEASUREMENTS AND MAIN RESULTS: The two study groups did not differ significantly in gestational age, birth weight, gender, duration of mechanical ventilatory support, surfactant therapy, duration of hospitalization, mortality rate, rate of bloodstream infection, or rate of colonization of tracheal aspirate, humidifier, and expiratory tube by microbes . The inspiratory tube was significantly less colonized in the 72-hr group as compared to the 24-hr group (p <.05) . The rate of VAP per 1000 ventilator days was not higher in the 72-hr group, compared with the 24-hr group (23.3 vs . 37.7; not significant) . Switching from a 24-hr to a 72-hr change policy would save our neonatal intensive care unit a yearly sum of $14,000 (US) . CONCLUSIONS: Extending the VC-change interval in premature infants from 24 hrs to 72 hrs is safe and cost-effective.

Medicina (Kaunas), 2003, 39(5), 460 - 3
{Mycoplasmal infection in patients with reactive arthritis and seronegative spondyloarthropaties}; Butrimiene I et al.; After investigation of 150 patients with reactive arthritis and undifferentiated spondyloarthropathies, inflammation signs of urogenital tract (uretritis in men and endocervicitis in women) were found in about 2/3 of patients . In those patients with reactive arthritis and undifferentiated spondyloarthropathies Ureaplazma urealyticum was found in more than 1/4 of patients, Mycoplasma hominis - in 1/10 of patients, and both microbes appeared in 1/10 of patients . Micoplasmal infection occurred in equal frequency in patients with reactive arthritis and undifferentiated spondyloarthropathies with a tendency to appear more in women than in men.

J Immunol, 2003 Jun 15, 170(12), 6151 - 7
The autoreactivity of anti-phosphorylcholine antibodies for atherosclerosis-associated neo-antigens and apoptotic cells; Shaw PX et al.; Abs specific for phosphorylcholine (PC) are known to contribute to the immune defense against a variety of microbial infections . To assess for other types of binding interactions, we performed surveys of anti-PC Abs of diverse biologic origins and structural diversity and demonstrated a common autoreactivity for oxidatively modified low density lipoprotein and other oxidation-specific structures containing PC-Ags . We also found that cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants . Whereas natural Abs to PC recognized cells at early stages of apoptosis, by contrast, an IgG anti-PC Ab, representative of a T cell-dependent response, recognized PC determinants primarily associated with late stages of apoptosis . Cumulatively, these results demonstrate a fundamental paradigm in which Abs from both the innate and the T cell-dependent tiers of the B cell compartment recognize a minimal molecular motif arrayed both on microbes and as neo-self Ags linked to atherosclerosis and autoimmune disease.

J Radiat Res (Tokyo), 2002 Dec, 43 Suppl, S7 - 12
Studies about space radiation promote new fields in radiation biology; Ohnishi T et al.; Astronauts are constantly exposed to space radiation of various types of energy with a low dose-rate during long-term stays in space . Therefore, it is important to determine correctly the biological effects of space radiation on human health . Studies about biological the effects at a low dose and a low dose-rate include various aspects of microbeams, bystander effects, radioadaptive responses and hormesis which are important fields in radiation biology . In addition, space radiations contain high linear energy transfer (LET) particles . In particular, neutrons may cause reverse effectiveness at a low dose-rate in comparison to ionizing radiation . We are also interested in p53-centered signal transduction pathways involved in the cell cycle, DNA repair and apoptosis induced by space radiations . We must also study whether the relative biological effectiveness (RBE) of space radiation is affected by microgravity which is another typical component in space . To confirm this, we must prepare centrifuge systems in an International Space Station (ISS) . In addition, we must prepare many types of equipment for space experiments in an ISS, because we cannot use conventional equipment from our laboratories . Furthermore, the research for space radiation might give us valuable information about the birth and evolution of life on the Earth . We can also realize the importance of preventing the ozone layer from depletion by the use of exposure equipment to sunlight in an ISS . For these reasons, we desire to educate space researchers of the next generation based on the consideration of the preservation of the Earth from research about space radiation.

J Infect Dis, 2003 Jun 15, 187 Suppl 2, S346 - 50
The immune response is initiated by dendritic cells via interaction with microorganisms and interleukin-2 production; Granucci F et al.; The immune system of vertebrate animals is characterized by the capacity to respond to disturbances . This function requires 2 different approaches . First, the immune system responds in a few hours to infectious agents (innate immunity) by recognizing molecular patterns typical of microorganisms (but absent in self-tissues) . Second, it mounts a late response that differentiates among different microbes, giving rise to memory (adaptive immunity) . In this context, dendritic cells (DCs) play a central role, becoming efficient stimulators of both innate and adaptive responses after microbial activation . Recent data generated by global transcriptional profiling of DCs after bacterial encounter are discussed, as are the unique DC functional plasticity and the central role of DC-derived interleukin-2 in priming early and late immune responses.

Nat Mater, 2003 Jul, 2(7), 487 - 92
X-ray microdiffraction study of growth modes and crystallographic tilts in oxide films on metal substrates; Budai JD et al.; The crystallographic texture of thin-film coatings plays an essential role in determining such diverse materials properties as wear resistance, recording density in magnetic media and electrical transport in superconductors . Typically, X-ray pole figures provide a macroscopically averaged description of texture, and electron backscattering provides spatially resolved surface measurements . In this study, we have used focused, polychromatic synchrotron X-ray microbeams to penetrate multilayer materials and simultaneously characterize the local structure, orientation and strain tensor of different heteroepitaxial layers with submicrometre resolution . Grain-by-grain microstructural studies of cerium oxide films grown on textured nickel foils reveal two distinct kinetic growth regimes on vicinal surfaces: ledge growth at elevated temperatures and island growth at lower temperatures . In addition, a combinatorial approach reveals that crystallographic tilting associated with these complex interfaces is qualitatively described by a simple geometrical model applicable to brittle films on ductile substrates . The sensitivity of conducting percolation paths to tilt-induced texture improvement is demonstrated.

Biotechnol Prog, 2003 May-Jun, 19(3), 899 - 907
Magnetophoretic cell sorting is a function of antibody binding capacity; McCloskey KE et al.; Antibody binding capacity (ABC) is a term representing a cell's ability to bind antibodies, correlating with the number of specific cellular antigens expressed on that cell . ABC allows magnetically conjugated antibodies to bind to the targeted cells, imparting a magnetophoretic mobility on each targeted cell . This enables sorting based on differences in the cell magnetophoretic mobility and, potentially, a magnetic separation based on the differences in the cell ABC values . A cell's ABC value is a particularly important factor in continuous magnetic cell separation . This work investigates the relationship between ABC and magnetic cell separation efficiency by injection of a suspension of immunomagnetically labeled quantum simply cellular calibration microbeads of known ABC values into fluid flowing through a quadrupole magnetic sorter . The elution profiles of the outlet streams were evaluated using UV detectors . Optimal separation flow rate was shown to correlate with the ABC of these microbeads . Comparing experimental and theoretical results, the theory correctly predicted maximum separation flow rates but overestimated the separation fractional recoveries.

Compend Contin Educ Dent, 2002 Nov, 23(11A), 1071 - 6, 1079-80, 1082
Clinical applications for host modulatory therapy; Ryan ME; A better understanding of the pathogenesis of periodontitis has resulted in pharmacotherapeutic advancements that address both the microbes and the host response, leading to improved management of this chronic progressive disease by the dental practitioner . The adjunctive use of host modulatory therapy can enhance therapeutic responses, slow the progression of the disease, and allow for more predictable management of patients, particularly in those patients at increased risk caused by factors beyond the reach of conventional therapeutic approaches . This article reviews the pathogenesis and risk factors associated with periodontitis and addresses, in detail, the concept and clinical use of host modulation as a therapeutic strategy.

J Allergy Clin Immunol, 2003 Jun, 111(6), 1370 - 4
Trichosporon pullulans infection in 2 patients with chronic granulomatous disease: an emerging pathogen and review of the literature; Moylett EH et al.; BACKGROUND: Chronic granulomatous disease is a genetically determined primary immunodeficiency disease in which phagocytic cells are unable to kill certain bacteria and fungi after ingestion . Manifestations include recurrent pyogenic infections caused by catalase-positive microbes . Trichosporon species are emerging as opportunistic agents that cause systemic disease in immunocompromised patients . Typically disease has been described in association with T beigelii in patients with secondary immunodeficiency, such as underlying malignancy . OBJECTIVE: The objective was to report the first 2 cases of T pullulans infection in 2 male children with chronic granulomatous disease . METHODS: The records of the 2 patients were reviewed . In addition, all cases of T pullulans infection reported in the English language literature are presented . RESULTS: This report brings to 7 the total number of cases of T pullulans reported and the first in patients with chronic granulomatous disease, one with invasive pneumonia and the other with an infected paronychium and localized cellulitis . In the 5 additional cases malignancy was the principal risk factor . CONCLUSION: T pullulans has rarely been reported as a fungal pathogen . The most prominent risk factor for the development of trichosporonosis is immunocompromise, most notably with neutropenia . Abnormally functioning neutrophils, such as with chronic granulomatous disease, may also predispose individuals to this opportunistic pathogen.

Reprod Nutr Dev, 2003 Jan-Feb, 43(1), 41 - 55
Effect of coconut oil and defaunation treatment on methanogenesis in sheep; Machmuller A et al.; The present study was conducted to evaluate in vivo the role of rumen ciliate protozoa with respect to the methane-suppressing effect of coconut oil . Three sheep were subjected to a 2 x 2 factorial design comprising two types of dietary lipids (50 g x kg(-1) coconut oil vs . 50 g x kg(-1) rumen-protected fat) and defaunation treatment (with vs . without) . Due to the defaunation treatment, which reduced the rumen ciliate protozoa population by 94% on average, total tract fibre degradation was reduced but not the methane production . Feeding coconut oil significantly reduced daily methane release without negatively affecting the total tract nutrient digestion . Compared with the rumen-protected fat diet, coconut oil did not alter the energy retention of the animals . There was no interaction between coconut oil feeding and defaunation treatment in methane production . An interaction occurred in the concentration of methanogens in the rumen fluid, with the significantly highest values occurring when the animals received the coconut oil diet and were subjected to the defaunation treatment . Possible explanations for the apparent inconsistency between the amount of methane produced and the concentration of methane-producing microbes are discussed . Generally, the present data illustrate that a depression of the concentration of ciliate protozoa or methanogens in rumen fluid cannot be used as a reliable indicator for the success of a strategy to mitigate methane emission in vivo . The methane-suppressing effect of coconut oil seems to be mediated through a changed metabolic activity and/or composition of the rumen methanogenic population.

Trends Microbiol, 2003 May, 11(5), 210 - 4
Neutrophil granulocytes--Trojan horses for Leishmania major and other intracellular microbes?
Laskay T, van Zandbergen G, Solbach W.
Polymorphonuclear neutrophil granulocytes (PMNs) possess numerous effector mechanisms to kill ingested pathogens as the first line of defence . However, several microorganisms evade intracellular killing in neutrophils, survive and retain infectivity . There is increasing evidence that several pathogens even multiply within neutrophils . Taking Leishmania major as a prototypic intracellular pathogen, we suggest an evasion strategy that includes the manipulation of PMNs in such a way that the pathogens are able to use the granulocytes as host cells . The ability to survive and maintain infectivity in PMNs subsequently enables these organisms to establish productive infection . These organisms can use granulocytes as Trojan horses before they enter their definitive host cells, the macrophages.

Int J Environ Health Res, 2003 Jun, 13 Suppl 1, S47 - 56
Epidemiological perspective of domestic and personal hygiene in India; Deodhar NS; When the application of epidemiology moves from mass phenomenon in a society or community to the specific family or individual level occurrences, new vistas unfold . The classical epidemiological triad, with its multi-mode influences and interactions, becomes modified as a result of several lifestyle factors coming into operation . It is well known that even under severely adverse climatic conditions, microbes are able to survive, and even propagate, if an appropriate micro-climate is encountered . This principle also applies to human beings . Many incidences of disease or ailments, occurrence or absence, can be traced to the home habitat, micro-ecosystem, human behaviour and lifestyles . Hygienic practices are largely a matter of behaviour and usually have biological and social origin . Human behaviour is influenced and determined by social traditions, customs and culture . Furthermore, factors such as health consciousness, practical knowledge of health sciences, motivation and concern for taking steps for promoting health and preventing disease, can change behaviour and make the lifestyle conducive to health . In a village or slum area, families live in more-or-less the same environment . However, in the event of an outbreak of a communicable disease, many escape the attack . While some experience frequent episodes of illness, others continue to live fairly healthily . Obviously, several social and cultural factors and associated human behaviours seem to make the difference between health and disease . This discussion examines the domestic and personal hygiene in its epidemiological perspective.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 643 - 7
The role of MyD88-like adapters in Toll-like receptor signal transduction; O'Neill LA; Signal-transduction pathways activated by Toll-like receptors (TLRs) have been the subject of intense investigation because of the key role played by TLRs in the recognition and elimination of microbes . Signalling is initiated by a domain termed the Toll/interleukin-1 (IL-1) receptor (TIR) domain that occurs on the cytosolic face of TLRs . This recruits, via homotypic interactions, adapter proteins that contain TIR domains . Three such adapter proteins have been discovered to date, and have been named MyD88, Mal {MyD88 adapter-like; also known as TIRAP (TIR domain-containing adapter protein)} and Trif (TIR-domain-containing adapter inducing interferon-beta) . Differences are emerging between TLRs in terms of which adapter is recruited by which TLR . This may lead to specificities in TLR signalling, with pathways being triggered that are specific for the elimination of the invading microbe . However, signals that separate Mal from MyD88 have yet to emerge, although biochemical differences between the two proteins imply that each will have a specific function.

Dig Dis Sci, 2003 May, 48(5), 976 - 85
Decreased gastric bacterial killing and up-regulation of protective genes in small intestine in gastrin-deficient mouse; Sun FJ et al.; Gastrin regulates gastric acid secretion, believed to be primarily responsible for killing ingested microbes . We examined gastric killing of gavaged E . coli in gastrin-deficient mice, which have decreased gastric acid production . Additionally, the expression of intestinal genes involved in epithelial protection were analyzed: the mucus layer glycoprotein muclin, the polymeric Ig receptor, trefoil factor 3, and small proline-rich protein 2a (sprr2a) . Gastric pH was 2.5 pH units greater in gastrin-deficient mice, and E . coli survival was increased greater than 20-fold at 10 min after gavage compared to control . Muclin and sprr2a gene expression were significantly increased (2.0- and 2.6-fold) in the intestine, and antibiotic treatment reversed these effects . In conclusion, reduced gastric acid secretion results in increased survival of ingested microorganisms in gastrin-deficient mice . Bacterial survival is associated with increased expression of muclin and sprr2a in the intestine, indicating that these genes play protective roles in the intestine.

J Insect Physiol, 2002 Aug, 48(8), 751 - 763
Could microbial symbionts of arthropod guts contribute significantly to nitrogen fixation in terrestrial ecosystems?
Nardi JB, Mackie RI, Dawson JO.
Fixed nitrogen is a limiting nutrient in most terrestrial ecosystems and has been assumed to be supplied almost entirely by free-living bacteria as well as by bacteria living in association with plants . The survival and growth of many arthropods on diets with extremely high carbon to nitrogen (C:N) ratios suggest that these arthropods are not obtaining sufficient nitrogen from their diets but must be obtaining additional nitrogen from some other source(s) . Estimates of N(2) fixation have suggested that symbiotic microbes of some arthropod hindguts could be obtaining this additional nitrogen as a result of nitrogen fixation . With the recent availability of antibody and nucleic acid probes, the presence of the enzyme that reduces dinitrogen gas to ammonia (nitrogenase) as well as the presence of its transcripts can be detected and localized with great sensitivity . A preliminary survey of a few detritivores indicates that nitrogen-fixing microbes of diverse forms are widespread in arthropod hindguts . In calculating nitrogen budgets, the possible contributions of nitrogen fixation by symbionts in arthropod guts, however, has been largely ignored . N(2) fixation in arthropod guts, with rates as high as 10-40 kg/ha/year being possible, may represent a significant contribution both to the growth of arthropods and to their ecosystem functions of processing carbon and nitrogen.

J Insect Physiol, 1998 Jul, 44(7-8), 645 - 651
Significance of methanogenic symbionts for development of the American cockroach, Periplaneta americana; Keddie BA et al.; The effects of suppression of methanogenesis with a drug, 2-bromoethanesulfonic acid (BES), on the hindgut ecosystem and development of the cockroach Periplaneta americana fed either low or high fiber diet were evaluated by measuring methane production, volatile fatty acids (VFA) concentrations in the hindgut, cockroach weight gain and development time (the length of nymphal period) . Methane production and VFA concentrations in the hindgut of cockroaches fed high fiber diet were significantly higher than those fed low fiber diet . Although BES treatment greatly reduced methane production, VFA concentrations in the hindgut, cockroach weight gain and development time were not significantly altered . These results indicate that methanogenic microbes are not essential for keeping low hydrogen pressure in the hindgut lumen, and normal cockroach development.

Curr Pharm Des, 2003, 9(14), 1115 - 20
Granulocyte colony-stimulating factor as the expecting sword for the treatment of severe sepsis; Murata A; Granulocyte colony-stimulating factor is a potent stimulator for neutrophils in the circulation as well as those progenitors to be increased and be grown in the bone marrow . Recombinant forms of this colony-stimulating factor, therefore, are widely using in the various kinds of clinical fields such as for the treatment of bone marrow suppression after cancer chemotherapy and for the treatment of myelodysplastic disorders . One of the physiological function of granulocyte colony-stimulating factor is to activate neutrophils enough to fight against invaded microbes and others, thus it is hoped to be used for the treatment of various kinds of infectious diseases even while the host has a normal number of circulating neutrophils and normal bone marrow . The future possible clinical usage of granulocyte colony-stimulating factor as for the immunomodulation against various kinds of insults to the human is reviewed.

J Acoust Soc Am, 2003 May, 113(5), 2820 - 33
Tongue-surface movement patterns during speech and swallowing; Green JR et al.; The tongue has been frequently characterized as being composed of several functionally independent articulators . The question of functional regionality within the tongue was examined by quantifying the strength of coupling among four different tongue locations across a large number of consonantal contexts and participants . Tongue behavior during swallowing was also described . Vertical displacements of pellets affixed to the tongue were extracted from the x-ray microbeam database . Forty-six participants recited 20 vowel-consonant-vowel (VCV) combinations and swallowed 10 ccs of water . Tongue-surface movement patterns were quantitatively described by computing the covariance between the vertical time-histories of all possible pellet pairs . Phonemic differentiation in vertical tongue motions was observed as coupling varied predictably across pellet pairs with place of articulation . Moreover, tongue displacements for speech and swallowing clustered into distinct groups based on their coupling profiles . Functional independence of anterior tongue regions was evidenced by a wide range of movement coupling relations between anterior tongue pellets . The strengths and weaknesses of the covariance-based analysis for characterizing tongue movement are considered.

Infect Immun, 2003 Jun, 71(6), 3337 - 42
Differential induction of interleukin-10 and interleukin-12 in dendritic cells by microbial toll-like receptor activators and skewing of T-cell cytokine profiles; Qi H et al.; Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of appropriate effector phenotypes accordingly . Microbial recognition and differentiation are mediated in part by pattern recognition receptors such as Toll-like receptors (TLRs), whereas the development of T-cell effector functions is critically dependent on DC-derived cytokines such as interleukin-12 (IL-12) and IL-10 . However, it is not entirely clear to what extent various microbial TLR activators could induce different functional states of DCs that favor different T-cell effector phenotypes . Toward a better understanding of this issue, we examined IL-10 and IL-12 production and T-cell-polarizing potentials of murine bone marrow-derived DCs after stimulation by three microbial TLR activators, namely, lipopolysaccharide (LPS), peptidoglycan (PGN), and zymosan . We found that the three stimuli induced drastically different profiles of IL-10 and IL-12 production in DCs . Further, these stimuli differentially conditioned CD40-dependent IL-10 and IL-12 production by DCs . Finally, LPS-, PGN-, and zymosan-stimulated DCs primed distinct T-cell cytokine profiles . Our results support the notion that microbe-specific information sensed through different TLRs by DCs is linked to differential Th priming through DC-derived cytokines.

Am J Ther, 2003 May-Jun, 10(3), 219 - 24
A swollen joint: why all the fuss?
Levine M, Siegel LB.
Acute arthritis may be a potential medical emergency . An infected joint causes rapid cartilaginous destruction and risk of future osteoarthritis . Prompt attention to the historical clues and potential causative organisms ensures appropriate therapy . Numerous microbes have been identified as the causative agent in septic arthritis, and various populations have distinct susceptibilities to these specific organisms . One broad classification of septic arthritis is differentiating gonococcal and nongonococcal organisms . This classification is important, as not only do the organisms differ, but the age of the patient and the portal of entry also differ . Aspiration of synovial fluid is paramount for proper diagnosis and management of septic arthritis . In addition, the timely administration of properly chosen antibiotic agents is essential for reducing morbidity and mortality.

Eur J Haematol, 2003 Jun, 70(6), 347 - 52
Investigation of megakaryocyte apoptosis in children with acute and chronic idiopathic thrombocytopenic purpura; Ucar C et al.; OBJECTIVE: Although the platelet destruction shows a primary role in the thrombocytopenia of idiopathic thrombocytopenic purpura (ITP), it has been demonstrated that impaired platelet production may also contribute to the severity of thrombocytopenia in ITP . The present study examined megakaryocyte apoptosis in bone marrow aspirates of children with acute and chronic ITP and investigated the role of megakaryocyte apoptosis in ITP pathophysiology . METHODS: Thirteen children diagnosed with acute ITP and eight children diagnosed with chronic ITP comprised the study group . Ten children, who were hospitalized for scoliosis operation but healthy otherwise, comprised the control group . In all children, megakaryocytes were isolated from the same amount of bone marrow aspirate samples using MACS CD61 MicroBeads (Miltenyl Biotec, Auburn, CA, USA) . Megakaryocyte apoptosis was studied with transferase-mediated d-UTP-bitin nick end-labeling method . RESULTS: Isolated megakaryocyte counts did not differ significantly between acute ITP, chronic ITP and control groups . The percentage of apoptotic megakaryocytes did not differ significantly between acute ITP group and control group and between chronic ITP group and control group . The percentage of apoptotic megakaryocytes in patients with chronic ITP was significantly lower than the patients with acute ITP . There was no correlation between the percentage of apoptotic megakaryocytes and platelet counts of the cases . CONCLUSIONS: Increased megakaryocytic apoptosis does not play a role in the pathogenesis of dysmegakaryopoiesis and impaired platelet production in children with ITP . Decreased megakaryocyte apoptosis in cases with chronic ITP may be due to suppression of megakaryocyte maturation, as the terminal phase of the megakaryocyte lifespan is characterized by the onset of apoptosis.

Radiats Biol Radioecol, 2003 Mar-Apr, 43(2), 223 - 6
Cell analysis with the new Leipzig high-energy ion nanoprobe; Osterreicher J et al.; The high-energy ion nanoprobe LIPSION at the University of Leipzig has been in operation since 1998 . The ultrastable, 3.5 MV SINLETRON accelerator supplies the H+ or He+ ion beam . A magnetic scanning system moves the focused beam across the sample . At present, a resolution of 41 +/- 4 nm in the low current mode and 300 nm at 5 pA can be achieved . The experimental chamber is equipped with electron-, energy dispersive X-ray-, and particle detectors . They can be used simultaneously to analyse the sample by means of PIXE (particle induced X-ray emission), RBS (Rutherford backscattering), and in the case of thin sections or monolayer samples STIM (scanning transmission ion microscopy) . A goniometer allows the application of channeling measurements in single crystals in combination with these methods . In contrast to previous publication describing microbeam facility at LIPSION, the current biomedical research has concentrated on microscopy and tomography on chondrocytes in pig cartilages and fixed single endothelial cells (HUVEC) . For the irradiation of single living cells, an external beam facility with irradiation platform, fast beamgate and mini-Petri dishes is under construction.

Respirology, 2003 Jun, 8(2), 140 - 8
The role of dendritic cells in immune regulation and allergic airway inflammation; Upham JW; Dendritic cells (DC) are potent antigen presenting cells that display an extraordinary capacity to present antigen to naive T-cells and initiate primary immune responses . In the context of the lung and upper airway it is clear that DC play a key role in the regulation of adaptive immune responses to inhaled antigen . DC are particularly sensitive to signals derived from microbes, allergens and the airway tissue microenvironment . By the nature of the signals they provide at the time of antigen presentation, DC can polarize naive T-cells into either T-helper type 1 (Th1) or Th2 effector cells, and are increasingly recognized as having a central role in the establishment of T-cell memory and peripheral immune tolerance . DC form a network within the upper airway and lung, and are rapidly recruited from the circulation in response to a variety of proinflammatory stimuli . Studies using animal models have highlighted the role of DC in both the initiation and maintenance of allergic airway inflammation . In early childhood, human DC are functionally immature, and this is thought to contribute to the development of allergic sensitization in those children who are genetically at risk for the development of atopy . Increased numbers of airway mucosal DC are found in both allergic rhinitis and asthma, while studies of blood-derived DC have emphasized important differences between the function of DC from atopic and normal individuals . This article reviews recent information on the involvement of DC in allergic airway disease, and the mechanisms by which DC could be exploited as targets for therapy in asthma and allergic rhinitis.

Lett Appl Microbiol, 2003, 36(6), 354 - 7
Methane oxidation and its coupled electron-sink reactions in ruminal fluid; Kajikawa H et al.; AIMS: This study was conducted to investigate the occurrence of methane oxidation in the rumen, and to identify the electron-sink reaction coupled to the oxidation if it occurred . METHODS AND RESULTS: Mixed ruminal microbes taken from sheep were incubated with 13CH4 . Oxidation of methane, estimated from the flux of 13C to CO2 and microbial cells, occurred, but represented only 0.2-0.5% of the methane produced . Methane oxidation was suppressed by the presence of oxygen, and was also inhibited by 2-bromoethane-sulphonate, and molybdate, but not by tungstate . CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Methane could be oxidized anaerobically in the rumen by reverse methanogenesis in consort with sulphate reduction.

Mol Ecol, 2003 Apr, 12(4), 1061 - 75
Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea; Russell JA et al.; To elucidate the co-evolutionary relationships between phloem-feeding insects and their secondary, or facultative, bacterial symbionts, we explore the distributions of three such microbes--provisionally named the R-type (or PASS, or S-sym), T-type (or PABS), and U-type--across a number of aphid and psyllid hosts through the use of diagnostic molecular screening techniques and DNA sequencing . Although typically maternally transmitted, phylogenetic and pairwise divergence analyses reveal that these bacteria have been independently acquired by a variety of unrelated insect hosts, indicating that horizontal transfer has helped to shape their distributions . Based on the high genetic similarity between symbionts in different hosts, we argue that transfer events have occurred recently on an evolutionary timescale . In several instances, however, closely related symbionts associate with related hosts, suggesting that horizontal transfer between distant relatives may be rarer than transmission between close relatives . Our findings on the prevalence of these symbionts within many aphid taxa, along with published observations concerning their effects on host fitness, imply a significant role of facultative symbiosis in aphid ecology and evolution.

J Appl Microbiol, 2003, 94(6), 1079 - 86
Taxonomic positioning of two biological control agents for plant diseases as Lysobacter enzymogenes based on phylogenetic analysis of 16S rDNA, fatty acid composition and phenotypic characteristics; Sullivan RF et al.; AIMS: To taxonomically position two bacterial strains conferring biological control activity towards plant diseases . METHODS AND RESULTS: Key phenotypic characteristics, including gliding motility and a high percentage of G + C content, indicated biocontrol strains N4-7 and C3 were essentially identical to those described for Lysobacter enzymogenes . Cellular fatty acid analysis confirmed a close relatedness of strains N4-7 and C3 to L . enzymogenes and a more distant relatedness to L . antibioticus . The 16S rDNA phylogenetic analysis revealed a distinct Lysobacter clade that included both strains within the gamma-proteobacteria . CONCLUSIONS: The combined taxonomic methods provide clear evidence that N4-7 and C3 should be grouped as strains of L . enzymogenes and not Stenotrophomonas maltophilia or a novel taxon . Phylogenetic analysis of 16S rDNA formed a Lysobacter clade that included several other environmentally diverse bacterial strains obtained from databases and confirmed relatedness of strains N4-7 and C3 to L . enzymogenes . SIGNIFICANCE AND IMPACT OF THE STUDY: Inclusion of N4-7 and C3 as strains of L . enzymogenes is among the first description of members of this genus as biocontrol agents of plant diseases . These results suggest that members of the Lysobacter group might provide a new source as plant-associated microbes that display biocontrol activity.

Proteomics, 2003 May, 3(5), 584 - 600
ProteomeWeb: a web-based interface for the display and interrogation of proteomes; Babnigg G et al.; The analysis of proteomes, i.e., the proteins expressed by biological organisms under a given set of conditions at a given time, requires separating complex protein mixtures into discrete protein components, measuring their relative abundances, and identifying the individual protein components . Many types of data are generated during the course of proteome analysis, including graphic images of the protein profiles, flat files containing numeric data, spreadsheets for assimilating numeric data, and relational database tables for integrating data from multiple experiments . As part of a project to describe the proteomes of microbes of interest to the U.S . Department of Energy, a World-Wide Web-based interface has been developed for the display of protein profiles generated by two-dimensional gel electrophoresis . The web interface is capable of obtaining protein identifications on the fly, interrogating the quantitative data in the context of available genome sequence information, and relating the proteome data to existing metabolic pathway databases . Analysis of protein expression profiles is expedited, providing the capability to efficiently determine the gene locations for proteins modulated in abundance in response to different growth conditions and to locate the positions of the proteins within specific metabolic pathways . The proteome of the archaeon Methanococcus jannaschii, a microbe for which the complete genome sequence is available, is used to demonstrate the capabilities of this evolving web interface .

Plant Physiol, 2003 May, 132(1), 146 - 53
Enhancement of plant-microbe interactions using a rhizosphere metabolomics-driven approach and its application in the removal of polychlorinated biphenyls; Narasimhan K et al.; Persistent organic pollutants, such as polychlorinated biphenyls (PCBs), are a global problem . We demonstrate enhanced depletion of PCBs using root-associated microbes, which can use plant secondary metabolites, such as phenylpropanoids . Using a "rhizosphere metabolomics" approach, we show that phenylpropanoids constitute 84% of the secondary metabolites exuded from Arabidopsis roots . Phenylpropanoid-utilizing microbes are more competitive and are able to grow at least 100-fold better than their auxotrophic mutants on roots of plants that are able to synthesize or overproduce phenylpropanoids, such as flavonoids . Better colonization of the phenylpropanoid-utilizing strain in a gnotobiotic system on the roots of flavonoid-producing plants leads to almost 90% removal of PCBs in a 28-d period . Our work complements previous approaches to engineer soil microbial populations based on opines produced by transgenic plants and used by microbes carrying opine metabolism genes . The current approach based on plant natural products can be applied to contaminated soils with pre-existing vegetation . This strategy is also likely to be applicable to improving the competitive abilities of biocontrol and biofertilization strains.

Biochem Biophys Res Commun, 2003 May 30, 305(2), 353 - 8
A novel cytomedical vehicle capable of protecting cells against complement; Yoshioka Y et al.; We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system . However, microencapsulated cells were not protected from humoral immune system . Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules . The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel . Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum . Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose . Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Apr, 11(2), 179 - 83
{Application of double immunomagnetic positive sorting to ex vivo expansion of marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria}; Xiao J et al.; Since flow cytometry was not feasible for sorting a huge amount of cells for clinical use, the method of double immunomagnetic positive sorting was used for selection of CD34(+)CD59(+) cells from bone marrow mononuclear cells in patients with paroxysmal nocturnal hemoglobinuria (PNH), which laid the groundwork for clinical ABMT/APBSCT of patients with PNH . Immunomagnetic positive selection was used for two times, the microbeads were removed from the CD34(+) cells selected firstly by means of overnight culture, then the sufficient CD34(+)CD59(+) cells were used for ex vivo expansion . The results showed that the survival, proliferation and colony-forming units of the selected CD34(+)CD59(+) cells by double immunomagnetic positive sorting had no significant difference as compared with that of CD34(+)CD59(+) cells selected by flow cytometry technique . It is suggested that the double immunomagnetic positive sorting promotes the use for separation and purification hematopoietic stem/progenitor cells and other cells with double or multiple markers cells for autologous hematopoietic stem cell transplantation in PNH patients.

Mol Plant Microbe Interact, 2003 Apr, 16(4), 306 - 14
Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula; Wulf A et al.; Significant changes in root morphology and physiology during arbuscular mycorrhiza (AM) development are likely to be controlled by specific gene expression pattern in the host plant . Until now, little was known about transcriptional changes which occur AM-exclusively; that is, they do not occur during other root-microbe associations, nor are they induced by improved phosphate nutrition . In order to identify such AM-exclusive gene inductions of Medicago truncatula, we used a pool of different RNA samples as subtractor population in a suppressive subtractive hybridization (SSH) experiment . This approach resulted in the identification of a number of new AM-regulated genes . None of these genes were expressed in nonmycorrhiza roots or leaves . Electronic data obtained by comparison of the cDNA sequences to expressed sequence tag (EST) sequences from a wide range of cDNA libraries in the M . truncatula EST database (Gene Index, MtGI) support the mycorrhiza specificity of the corresponding genes, because sequences in the MtGI that were found to match the identified SSH-cDNA sequences originated exclusively from AM cDNA libraries . The promoter of one of those genes, MtGst1, showing similarities to plant glutathione-S-transferase (GST) encoding genes, was cloned and used in reporter gene studies . In contrast to studies with the potato GST gene PRP, MtGst 1 promoter activity was detected in all zones of the root cortex colonized by Glomus intraradices, but nowhere else.

Mol Biochem Parasitol, 2003 May, 128(2), 217 - 28
Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania; Robinson KA et al.; Approaches which eliminate mRNA expression directly are ideally suited for reverse genetics applications in eukaryotic microbes which are asexual diploids, such as the protozoan parasite Leishmania . RNA interference (RNAi) approaches have been successful in many species, including the related parasite Trypanosoma brucei . For RNAi tests in Leishmania, we developed improved protocols for transient and stable DNA transfection, attaining efficiencies of up to 25 and 3%, respectively . This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition gives a strong lethal phenotype in trypanosomatids . However, transient or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-loop construct and GFP to monitor transfection resulted in no effect on parasite morphology, growth or tubulin expression in Leishmania major or L . donovani . Transient transfection of a 24-nucleotide double-stranded alpha-tubulin siRNA also had no effect . Similar results were obtained in studies targeting an introduced GFP gene with a GFP stem-loop construct . These data suggest that typical RNAi strategies may not work effectively in Leishmania, and raise the possibility that Leishmania is naturally deficient for RNAi activity, like Saccharomyces cerevisae . The implications to parasite biology, gene amplification, and genetic analysis are discussed.

Biomacromolecules, 2003 May-Jun, 4(3), 793 - 8
Injectable polymeric microspheres with X-ray visibility . Preparation, properties, and potential utility as new traceable bulking agents; Saralidze K et al.; The copolymer of methyl methacrylate (MMA) and 2-{2',3',5'-triiodobenzoyl}oxoethyl methacrylate (1), ratio 3:1 (mass:mass), was prepared via a free-radical polymerization in bulk . The copolymer (M(w) = 97.8 kD and M(n) = 41.5 kD) was dissolved in chloroform and subsequently transformed into beads with a diameter in the micrometer range, using a solvent evaporation technique . The resulting microbeads were characterized by different techniques, including NMR spectroscopy, differential scanning calorimetry, gel permeation chromatography, and scanning electron microscopy . The latter technique was used as the basis for statistical analysis of the bead size . Typically, an average diameter of 96 microm and a standard deviation of 21 microm were obtained . The beads were also subjected to some preliminary tests regarding cytotoxicity . The copolymer of MMA and 1 contains covalently bound iodine . Therefore, the material is intrinsically radiopaque, i.e., capable of absorbing X-radiation while no contrast additive is needed . Our interest in these microspheres stems primarily from their possible utility as injectable and afterward traceable (radiopaque) bulking agents, e.g., for use in urology for the treatment of female stress incontinence due to sphincter deficiency . As a first test into this direction, a sample of the microbeads was mixed with ethylene glycol, and the resulting suspension was studied with respect to injectability and radiopacity . The results suggest that the radiopaque microbeads may provide access to improved bulking agents . Further modification of the surface may be necessary in order to suppress the migratory aptitude of the radiopaque polymeric microspheres in vivo.

Microb Ecol, 2003 Jul, 46(1), 134 - 44 Epub 2003 May 13.
Root-parasitic nematodes enhance soil microbial activities and nitrogen mineralization; Tu C et al.; Obligate root-parasitic nematodes can affect soil microbes positively by enhancing C and nutrient leakage from roots but negatively by restricting total root growth . However, it is unclear how the resulting changes in C availability affect soil microbial activities and N cycling . In a microplot experiment, effects of root-parasitic reniform nematodes ( Rotylenchulus reniformis) on soil microbial biomass and activities were examined in six different soils planted with cotton . Rotylenchulus reniformis was introduced at 900 nematodes kg(-1) soil in May 2000 prior to seeding cotton . In 2001, soil samples were collected in May before cotton was seeded and in November at the final harvest . Extractable C and N were consistently higher in the R . reniformis treatments than in the non-nematode controls across the six different soils . Nematode inoculation significantly reduced microbial biomass C, but increased microbial biomass N, leading to marked decreases in microbial biomass C:N ratios . Soil microbial respiration and net N mineralization rates were also consistently higher in the nematode treatments than in the controls . However, soil types did not have a significant impact on the effects of nematodes on these microbial parameters . These findings indicate that nematode infection of plant roots may enhance microbial activities and the turnover of soil microbial biomass, facilitating soil N cycling . The present study provides the first evidence about the direct role of root-feeding nematodes in enhancing soil N mineralization.

Microbes Infect, 2003 Apr, 5(5), 429 - 37
Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants?
Panstruga R, Schulze-Lefert P.
Human diseases like AIDS, malaria, and pneumonia are caused by pathogens that corrupt host chemokine G-protein coupled receptors for molecular docking . Comparatively, little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes . Here, we review potential analogies between human chemokine receptors and the plant seven-transmembrane MLO protein, a candidate serving a dual role as docking molecule and defence modulator for the phytopathogenic powdery mildew fungus.

J Biol Chem, 2003 Aug 1, 278(31), 28443 - 54 Epub 2003 May 07.
Elucidation of molecular events leading to neutrophil apoptosis following phagocytosis: cross-talk between caspase 8, reactive oxygen species, and MAPK/ERK activation; Zhang B et al.; Phagocytosis of complement-opsonized targets is a primary function of neutrophils at sites of inflammation, and the clearance of neutrophils that have phagocytosed microbes is important for the resolution of inflammation . Our previous work suggests that phagocytosis leads to rapid neutrophil apoptosis that is inhibited by antibody to the beta2 integrin, Mac-1, and requires NADPH oxidase-derived reactive oxygen species (ROS) generated during phagocytosis . Here we report that phagocytosis-induced cell death (PICD) does not occur in Mac-1-deficient murine neutrophils, suggesting that PICD proceeds through a bona fide Mac-1-dependent pathway . A sustained, intracellular oxidative burst is associated with PICD . Furthermore, PICD does not require traditional death receptors, Fas, or tumor necrosis factor (TNF) receptor . TNF but not Fas synergizes with phagocytosis to enhance significantly PICD by increasing the oxidative burst, and this is Mac-1-dependent . Phagocytosis-induced ROS promote cleavage/activation of caspases 8 and 3, key players in most extrinsic ("death receptor") mediated pathways of apoptosis, and caspases 8 and 3 but not caspase 9/mitochondria, are required for PICD . This suggests that ROS target the extrinsic versus the intrinsic ("stress stimulus") apoptotic pathway . Phagocytosis also triggers a competing MAPK/ERK-dependent survival pathway that provides resistance to PICD likely by down-regulating caspase 8 activation . The anti-apoptotic factor granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhances ROS generation associated with phagocytosis . Despite this, it completely suppresses PICD by sustaining ERK activation and inhibiting caspase 8 activation in phagocytosing neutrophils . Together, these studies suggest that Mac-1-mediated phagocytosis promotes apoptosis through a caspase 8/3-dependent pathway that is modulated by NADPH oxidase-generated ROS and MAPK/ERK . Moreover, TNF and GM-CSF, likely encountered by phagocytosing neutrophils at inflammatory sites, exploit pro-(ROS) and anti-apoptotic (ERK) signals triggered by phagocytosis to promote or suppress PICD, respectively, and thus modulate the fate of phagocytosing neutrophils.

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