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 dres