Bacteria

Nature, 2001 Mar 1, 410(6824), 77 - 81
Isotopic evidence for microbial sulphate reduction in the early Archaean era; Shen Y et al.; Sulphate-reducing microbes affect the modern sulphur cycle, and may be quite ancient, though when they evolved is uncertain . These organisms produce sulphide while oxidizing organic matter or hydrogen with sulphate . At sulphate concentrations greater than 1 mM, the sulphides are isotopically fractionated (depleted in 34S) by 10-40/1000 compared to the sulphate, with fractionations decreasing to near 0/1000 at lower concentrations . The isotope record of sedimentary sulphides shows large fractionations relative to seawater sulphate by 2.7 Gyr ago, indicating microbial sulphate reduction . In older rocks, however, much smaller fractionations are of equivocal origin, possibly biogenic but also possibly volcanogenic . Here we report microscopic sulphides in approximately 3.47-Gyr-old barites from North Pole, Australia, with maximum fractionations of 21.1/1000, about a mean of 11.6/1000, clearly indicating microbial sulphate reduction . Our results extend the geological record of microbial sulphate reduction back more than 750 million years, and represent direct evidence of an early specific metabolic pathway--allowing time calibration of a deep node on the tree of life.

Mutat Res, 2001 Mar 1, 474(1-2), 159 - 68
Polar alteration of short tandem repeats (STRs) in mammalian cells; Suzuki A et al.; Instability of short tandem repeats (STRs) in DNA during replication is observed in all organisms examined, and is causatively involved in various human diseases . We explore the mechanisms involved in instability by examining length changes occurring during the replication of {(CA)(20)TA}(n) and {(CAG)(20)TAG}(n), in human cells . We show that the majority of alterations consist of an insertion or deletion of one repeat unit, and base substitutions or length changes involving many repeat units are rare . We also show that length changes of two-tract STRs are biased toward the 3'-end of the repeat tract, in reference to lagging strand synthesis . There are some differences between our observations and previous observations in microbes, e.g . the orientation effect was not observed in this study . The results of this study are discussed in terms of the molecular mechanisms leading to alterations in repeat tracts.

Microbiol Mol Biol Rev, 2001 Mar, 65(1), 106 - 18
Biosynthesis of polyketides in heterologous hosts; Pfeifer BA et al.; Polyketide natural products show great promise as medicinal agents . Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases . A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest . To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis . Here we review the state of the art as well as the difficulties associated with heterologous polyketide production . In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli . Future directions for this relatively new but growing technological opportunity are also discussed.

J Immunol, 2001 Mar 15, 166(6), 4237 - 43
Environmental modulation of autoimmune arthritis involves the spontaneous microbial induction of T cell responses to regulatory determinants within heat shock protein 65; Moudgil KD et al.; Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases . Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65) . We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65 . The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water . Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag . Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA . These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats . These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.

Br J Cancer, 2001 Mar 2, 84(5), 674 - 9
Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts; Belyakov OV et al.; Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment . Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes . In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts . We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles . Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha-particles) . The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 micrometer and a detection and counting efficiency of greater than 99% . When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells . The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell . Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish . Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish . This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved .

Anal Biochem, 2001 Mar, 290(2), 214 - 20
Miniaturization of a hepatitis C virus RNA polymerase assay using a -102 degrees C cooled CCD camera-based imaging system; Zheng W et al.; Innovations in detection technologies have allowed us to develop a novel assay in 1536-well plate format and assess the advantages of screen miniaturization compared with conventional high-throughput compound screening in 96- or 384-well plates . An HCV RNA polymerase assay has been miniaturized in 1536-well plates by using a new detection technology known as LEADseeker homogeneous imaging system . It uses a -102 degrees C cooled charge-coupled device (CCD) camera and newly designed scintillation proximity microparticles . The miniaturized assay used europium-doped streptavidin-coated yttrium oxide (YO(x)) or polystyrene (PS) microspheres to capture biotin-labeled {(3)H}RNA product transcripts . Beads in proximity to the radioisotope convert the emitted beta(-) particles into photons having wavelengths in the red region of the visible spectrum, optimal for detection by the CCD camera . Because the camera collects light from all wells of the plate simultaneously, 1536-well plates are imaged as rapidly as 384-well plates, on the order of 10 min per plate . The assay has a signal to background of approximately 20-fold, satisfactory for high-throughput robotics screening . The enzyme kinetics and potency of a known inhibitor were similar to those obtained from the conventional assay using scintillation proximity assay (SPA) beads and a scintillation plate counter . Furthermore, the newly developed microbeads (emitting at 610 to 620 nm) are less prone to quenching effects caused by yellow-colored compounds, than conventional SPA beads or scintillation fluid (emitting at 400 to 480 nm region) . Thus, the LEADseeker imaging system is a useful new tool for miniaturization of assays for high-throughput screening .

Zh Mikrobiol Epidemiol Immunobiol, 2001 Jan-Feb, (1), 104 - 8
{Effect of microalgae on viability of microorganisms in the natural and artificial environment}; Gorobets OB et al.; The data of literature on complicated relationships between microorganisms and microalgae in algobacterial associations with microalgae often playing the leading role are analyzed . Under these conditions inhibiting and stimulating substances synthesized by microalgae, as well as their nutritional value, may have an essential impact on the state of the bacterial population . Apparently that microalgae may become the main substrate in the development of new culture media (ecologically pure culture media, media for the reversion of the noncultured forms of bacteria into the vegetative state, media for the prolonged preservation of microbes in the noncultured form).

Appl Microbiol Biotechnol, 2001 Jan, 55(1), 1 - 9
Microbial formation, biotechnological production and applications of 1,2-propanediol; Bennett GN et al.; This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes . 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized . One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol . The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol . The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product . The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway . Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose . The optimization of these strains may yield microbial processes for the production of this widely used chemical.

Prikl Biokhim Mikrobiol, 2001 Jan-Feb, 37(1), 123 - 8
{Rapid assay for the assessment of a potential of chemical biocides to microbial destructors of industrial materials}; Novikov IA et al.; A colorimetric rapid assay for estimating the biocide potential of various chemicals towards metal biocorrosive and petroleum product degrading microbes was developed based on the reducing potential of live microbial cell . A water-soluble organic redox indicator, blue in the oxidized form and pink in the reduced form, was used as an indicator of the reducing potential of microbial cells . Once added to a suspension of vital microbial cells, it was reduced and changed in color . A good correlation between the results of this assay and viability control was obtained by employing surfactants and heavy metal ions.

Nature, 2001 Feb 22, 409(6823), 1092 - 101
Life in extreme environments; Rothschild LJ et al.; Each recent report of liquid water existing elsewhere in the Solar System has reverberated through the international press and excited the imagination of humankind . Why? Because in the past few decades we have come to realize that where there is liquid water on Earth, virtually no matter what the physical conditions, there is life . What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring 'extremophiles' . This realization, coupled with new data on the survival of microbes in the space environment and modelling of the potential for transfer of life between celestial bodies, suggests that life could be more common than previously thought . Here we examine critically what it means to be an extremophile, and the implications of this for evolution, biotechnology and especially the search for life in the Universe.

Folia Biol (Praha), 2001, 47(1), 36 - 9
Analysis of paternal alleles in nucleated red blood cells enriched from maternal blood; Hromadnikova I et al.; The purpose of our study was to identify paternal alleles in NRBC enriched from maternal peripheral blood for detection of the presence of foetal cells in the maternal circulation and to establish a reliable non-invasive method which should allow following genetic testing . For enrichment of foetal cells from peripheral maternal blood we combined Ficoll-Paque density gradient centrifugation and MACS . Maternal leukocytes were firstly depleted using anti-CD14 and anti-CD45 microbeads . NRBC were sorted from the CD14-/CD45- fraction by positive selection using CD71 microbeads . Paternal alleles in the CD14-/CD45-/CD71+ fraction were indicated by the PCR method using HLA (DRB1, DQB1, DQA1) and Polymarker System (LDLR, GYPA, HBGG, D7S8, GC) as genetic markers . Different paternal alleles of studied 8 loci were detected in 13 out of 19 samples of cells enriched from maternal peripheral blood between the 13th and 36th week of gestation . Our results demonstrate that foetal cells enriched from maternal peripheral blood may be used as a source of foetal DNA for prenatal diagnosis, paternity testing and other application.

Plant Cell Physiol, 2001 Feb, 42(2), 129 - 37
An arabinogalactan protein(s) is a key component of a fraction that mediates local intercellular communication involved in tracheary element differentiation of zinnia mesophyll cells; Motose H et al.; Local intercellular communication is involved in tracheary element (TE) differentiation of zinnia (Zinnia elegans L.) mesophyll cells and mediated by a proteinous macromolecule, which was designated xylogen . To characterize and isolate xylogen, a bioassay system to monitor the activity of xylogen was developed, in which mesophyll cells were embedded in microbeads of agarose gel at a low (2.0-4.3x10(4) cells ml(-1)) or high density (8.0-9.0x10(4) cells ml(-1)) and microbeads of different cell densities were cultured together in a liquid medium to give a total density of 2.1-2.5x10(4) cells ml(-1) . Without any additives, the frequency of TE differentiation was much smaller in the low-density microbeads than in the high-density microbeads . This low level of TE differentiation in the low-density microbeads was attributable to the shortage of xylogen . When cultures were supplemented with conditioned medium (CM) prepared from zinnia cell suspensions undergoing TE differentiation, the frequency of TE differentiation in the low-density microbeads increased remarkably, indicating the activity of xylogen in the CM . The xylogen activity in CM was sensitive to proteinase treatments . Xylogen was bound to galactose-specific lectins such as Ricinus communis agglutinin and peanut agglutinin, and precipitated by beta-glucosyl Yariv reagent . These results indicate that xylogen is a kind of arabinogalactan protein.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2164 - 9
Truncated hexa-octahedral magnetite crystals in ALH84001: presumptive biosignatures; Thomas-Keprta KL et al.; McKay et al . {(1996) Science 273, 924-930} suggested that carbonate globules in the meteorite ALH84001 contained the fossil remains of Martian microbes . We have characterized a subpopulation of magnetite (Fe(3)O(4)) crystals present in abundance within the Fe-rich rims of these carbonate globules . We find these Martian magnetites to be both chemically and physically identical to terrestrial, biogenically precipitated, intracellular magnetites produced by magnetotactic bacteria strain MV-1 . Specifically, both magnetite populations are single-domain and chemically pure, and exhibit a unique crystal habit we describe as truncated hexa-octahedral . There are no known reports of inorganic processes to explain the observation of truncated hexa-octahedral magnetites in a terrestrial sample . In bacteria strain MV-1 their presence is therefore likely a product of Natural Selection . Unless there is an unknown and unexplained inorganic process on Mars that is conspicuously absent on the Earth and forms truncated hexa-octahedral magnetites, we suggest that these magnetite crystals in the Martian meteorite ALH84001 were likely produced by a biogenic process . As such, these crystals are interpreted as Martian magnetofossils and constitute evidence of the oldest life yet found.

Curr Opin Gastroenterol, 2001 Mar, 17(2), 171 - 176
Nutrition and the mucosal immune system; Cunningham-Rundles S; Gut-associated lymphoid tissue is the dominant site for the initiation of mucosal immune response . Mucosal immunity depends on regulatory signals; nutritional elements, including fats, amino acids, and micronutrients, are critical cofactors for these signals . Nutrients specifically affect lymphocyte influx and migration, mononuclear cell activation, and the differentiated expression of immune response . The molecular basis of nutrient action has been shown to involve effects on receptor regulation, adhesion molecule expression, and the pattern of cytokine production . The gastrointestinal mucosal immune system is the major site for host interaction with microbes and provides a barrier against systemic access for food antigens and microbes . Nutrient metabolism has unique and direct impact on the host defense system of gut-associated lymphoid tissue and therefore has potential for widely disseminated impact on systemic immune response.

Br J Ophthalmol, 2001 Mar, 85(3), 336 - 40
A comparison of cyst age and assay method of the efficacy of contact lens disinfectants against Acanthamoeba; Kilvington S et al.; AIMS: (i) To determine effect of Acanthamoeba cyst age, method of production, and (ii) to assay technique on the efficacy of multipurpose solutions (MPS) and hydrogen peroxide based contact lens disinfectants . (iii) To establish if MPS can remove mature cysts from contact lenses according to the ISO/DIS 14729 regimen test for microbe removal . METHODS: Immature and mature cysts of A polyphaga were tested against the MPS Opti-Free express and the hydrogen peroxide based solutions Oxysept 1Step and Oxysept 1 using two assay methods . Simulated patient regimen testing was performed with the Opti-Free express and Complete using mature cysts inoculated on to group I or group IV lenses . RESULTS: Immature cysts were sensitive to disinfection by all solutions . No killing was observed with mature cysts with Opti-Free express, while immature cysts yielded a 1-2 log reduction in viability . Oxysept 1Step gave a 1.1 (SD 0.3) log reduction in mature cysts after 6 hours . Oxysept 1 gave a 2.4 (0.3) log reduction in mature cysts after 4 hours and a 3.8 (0.5) log reduction after 6 hours . Patient regimen testing using Opti-Free express and Complete resulted in no recovery of viable mature cysts from the contact lenses or from the soaking solutions . CONCLUSION: Cyst age but not method of production used in this study influences the efficacy of contact lens disinfectants against Acanthamoeba . MPS are effective in removing cysts from contact lens surfaces and may have a role in the prevention of acanthamoeba keratitis.

Ann Biomed Eng, 2001 Jan, 29(1), 1 - 8
Controlled cell deformation produces defined areas of contact between cells and ligand-coated surfaces; Patrick SM et al.; A method which allows precise control of the time of initiation and the area of contact of T cells with immobilized ligands has been developed . Cells are trapped in an asymmetric film that can be quantitatively thinned by reducing the film's capillary pressure . Ligands adsorbed to the base of the apparatus are forced into close contact with the cells as the air-liquid interface is drawn down . Using interference microscopy and microbeads to indicate the film height, the amount of thinning can be controlled to within 1 microm . In this study, this system was used to produce contact areas of 182 and 356 microm2 between T cells and anti-CD3 coated surfaces . These contact areas were measured using fluorescent dye exclusion microscopy . This apparatus can be used for quantitative studies of T cell activation, as is reported in Patrick et al., J . Immunol . Method . 24:97-108, 2000.

J Chromatogr A, 2001 Feb 9, 909(1), 53 - 60
Detection of the cyclic nitramine explosives hexahydro-1,3,5-trinitro- 1,3,5-triazine (RDX) and octahydro- 1,3,5,7-tetranitro- 1,3,5,7-tetrazine (HMX) and their degradation products in soil environments; Groom CA et al.; The cyclic nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine (HMX) were examined in field and microcosm soil samples to determine their patterns of degradation and environmental fates . A number of analytical techniques, including solid-phase microextraction with on-fiber derivatization, gas chromatography-mass spectrometry, gas chromatography with electron-capture detection, liquid chromatography-mass spectrometry, and micellar electrokinetic chromatography were required for the analyses . Two different classes of intermediates were detected, both of which lead ultimately to the formation of nitrous oxide (N2O) and carbon dioxide (CO2) . The first class was identified as the nitroso derivatives formed by the sequential reduction of -NO2 functional groups . The second class of intermediates, which was favored at higher humidities and in the presence of anaerobic sludge amendments, consisted of ring cleavage products including bis-(hydroxymethyl)-nitramine and methylenedinitramine . Rye-grass (Lolium perenne) present in field samples was found to extract and accumulate HMX from soil without further degradation . In all cases (excepting the plant samples), the indigenous microbes or amended domestic anaerobic sludge consortia degraded the cyclic nitramine explosives eventually to produce N2O and CO2.

Proc R Soc Lond B Biol Sci, 2001 Feb 7, 268(1464), 303 - 9
Microbial diversity, producer-decomposer interactions and ecosystem processes: a theoretical model; Loreau M; Interactions between the diversity of primary producers and that of decomposers--the two key functional groups that form the basis of all ecosystems--might have major consequences on the functioning of depauperate ecosystems . I present a simple ecosystem model in which primary producers (plants) and decomposers (microbes) are linked through material cycling . The model considers a diversity of plant organic compounds and a diversity of microbial species . Nutrient recycling efficiency from organic compounds to decomposers is then the key parameter that controls ecosystem processes (primary productivity, secondary productivity, producer biomass and decomposer biomass) . The model predicts that microbial diversity has a positive effect on nutrient recycling efficiency and ecosystem processes through either greater intensity of microbial exploitation of organic compounds or functional niche complementarity, much like in plants . Microbial niche breadth and overlap should not affect ecosystem processes unless they increase the number of organic compounds that are decomposed . In contrast, the model predicts that plant organic compound diversity can only have a negative effect or, at best, no effect on ecosystem processes, at least in a constant environment . This creates a tension between the effects of plant diversity and microbial diversity on ecosystem functioning, which may explain some recent experimental results.

Nature, 2001 Feb 1, 409(6820), 630 - 3
Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo; Grill SW et al.; Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development . In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase . The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear . Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo . To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin . We show that pulling forces external to the spindle act on the two spindle poles . A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos . We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior-posterior embryonic axis . Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour . Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.

Cell Mol Life Sci, 1999 Nov 30, 56(9-10), 742 - 54
Antibiotic resistance in microbes; Mazel D et al.; The treatment of infectious disease is compromised by the development of antibiotic-resistant strains of microbial pathogens . A variety of biochemical processes are involved that may keep antibiotics out of the cell, alter the target of the drug, or disable the antibiotic . Studies have shown that resistance determinants arise by either of two genetic mechanisms: mutation and acquisition . Antibiotic resistance genes can be disseminated among bacterial populations by several processes, but principally by conjugation . Thus the overall problem of antibiotic resistance is one of genetic ecology and a better understanding of the contributing parameters is necessary to devise rational approaches to reduce the development and spread of antibiotic resistance and so avoid a critical situation in therapy--a return to a pre-antibiotic era.

Australas Phys Eng Sci Med, 2000 Sep, 23(3), 88 - 93
Monte Carlo calculation for microplanar beam radiography; Company FZ et al.; In radiography the scattered radiation from the off-target region decreases the contrast of the target image . We propose that a bundle of collimated, closely spaced, microplanar beams can reduce the scattered radiation and eliminate the effect of secondary electron dose, thus increasing the image dose contrast in the detector . The lateral and depth dose distributions of 20-200 keV microplanar beams are investigated using the EGS4 Monte Carlo code to calculate the depth doses and dose profiles in a 6 cm x 6 cm x 6 cm tissue phantom . The maximum dose on the primary beam axis (peak) and the minimum inter-beam scattered dose (valley) are compared at different photon energies and the optimum energy range for microbeam radiography is found . Results show that a bundle of closely spaced microplanar beams can give superior contrast imaging to a single macrobeam of the same overall area.

Ann Med, 2000 Dec, 32(9), 585 - 7
The future of fluoroquinolones; Drlica K; The mutant prevention concentration (MPC) is a new measure of antibiotic potency above which a microbe must attain two concurrent resistance mutations for growth . For some C-8-methoxy fluoroquinolone-pathogen combinations, the value of MPC is below human serum drug concentration achieved with standard doses . Although untested clinically, such a low value of MPC, coupled with high serum concentration, should allow these fluoroquinolones to restrict severely the selection of resistant mutants when used as monotherapy . Compounds that cannot meet the MPC-pharmacokinetic criterion will enrich resistant mutants unless they are a part of combination therapy . Separation of fluoroquinolones into groups suitable for monotherapy or for combination therapy, followed by appropriate adminstration, may help extend the lifespan of the fluoroquinolones.

J Vet Med B Infect Dis Vet Public Health, 2000 Dec, 47(10), 727 - 37
Relationship between the amounts of antibodies to Actinobacillus pleuropneumoniae serotype 2 detected in blood serum and in fluids collected from muscles of pigs; Wallgren P et al.; An indirect ELISA method, previously used to detect antibodies to Actinobacillus pleuropneumoniae serotype 2 in serum of pigs, was further developed aiming to measure antibodies to the microbe in muscle fluids . Serum and muscle fluid were collected from Specific Pathogen Free (SPF) pigs as well as from SPF pigs challenged with A . pleuropneumoniae which were either treated with effective antibiotics or left as infected controls . The antibody responses measured in serum correlated well to the clinical signs of respiratory disease observed and to pathological lesions found at necropsy performed 17 days post-infection . The amounts of antibodies monitored in serum and in muscle fluid collected from the diaphragm and the thigh, respectively, were compared . Higher concentrations of antibodies were assessed in serum than in diaphragm fluid, which in turn contained more antibodies per ml than fluid collected from the thigh . The amount of antibodies to A . pleuropneumoniae measured in fluid from the diaphragm diluted 1/50 correlated well with the quantity measured in serum diluted 1/1000 (r2 = 0.87; P < 0.001) . When validated by using serum antibody responses as a standard, the specificity of the ELISA employed in fluid from the diaphragm was found to be 100% . The sensitivity was determined to be 88% when calculated on seropositive pigs (A450 = 0.3 in serum diluted 1/1000) . That figure increased to 97% if calculated on pigs expressing pronounced amounts of serum antibodies (A450 > or = 0.5).

J Neuropathol Exp Neurol, 2001 Jan, 60(1), 33 - 48
Effects of sodium and chloride on neuronal survival after neurite transection; Rosenberg LJ et al.; An in vitro investigation was undertaken to study the roles of Na+ and Cl- in mammalian spinal cord (SC) neuron deterioration and death after injury involving physical disruption of the plasma membrane . Individual SC neurons in monolayer cultures were subjected to UV laser microbeam transection of a primary dendrite . Neurons lesioned in modified ionic environments (MIEs) where 50%-75% of the NaCl was replaced with sucrose had higher survival (65%-75%) than neurons lesioned in medium with normal (125 mM) NaCl (28%; p < 0.001) . Subsequent experiments found a comparable increase in lesioned neuron survival in MIEs in which only Na+ was replaced with specific ionic substitutes; however, replacement of Cl- was not protective . Electron microscope examinations of neurons fixed <16 min after lesioning showed a dramatic decrease in vesiculation of the smooth endoplasmic reticulum and Golgi apparatus in the low NaCl or low Na+ MIEs . It is hypothesized that Na+ entry after membrane disruption may stimulate elevation of {Ca+2}i leading to ultrastructural disruption and death of injured neurons . The results of these studies suggest that a low NaCl MIE may be useful as an irrigant to limit damage spread and cell death within CNS tissues during surgery or after trauma.

Br Poult Sci, 2000 Dec, 41(5), 575 - 83
Investigation of hygiene aspects during air chilling of poultry carcases using a model rig; Allen VM et al.; 1 . An experimental rig, designed and built to simulate conditions found in commercial poultry chilling systems, was used to investigate the effects of varying air temperature and chilling duration, and the effect of chlorinated water sprays, on the microbial load present on the skin and in the body cavity of freshly eviscerated poultry carcases; deep muscle and skin temperatures were monitored during chilling at three different temperatures . 2 . During dry chilling for 2 h, total viable microbe counts (TVC) and counts of coliforms and pseudomonads from the body cavity fell by between half and one log unit; smaller reductions were observed in samples from the breast skin . 3 . The situation changed when chlorinated water sprays (50, 100 or 250 ppm available chlorine) were applied for the first hour of chilling; spraying carcases enhanced the reduction in numbers on the skin; the effect was most pronounced with 250 ppm chlorine; conversely in the body cavity, the general effects of sprays was to increase contamination by up to one log unit . 4 . There was no evidence that sprays increased the rate of chilling . 5 . When carcases were held overnight in the rig at 11 degrees C after chilling, microbe counts on dry-chilled carcases remained stable, but increased on carcases that had been sprayed with chlorinated water.

J Behav Med, 2000 Dec, 23(6), 519 - 29
Serum cholesterol concentrations and mood states in violent psychiatric patients: an experience sampling study; Hillbrand M et al.; The well-documented negative association between serum cholesterol and aggressive behavior has led Kaplan to propose a cholesterol-serotonin hypothesis of aggression . According to this hypothesis, low dietary cholesterol intake leads to depressed central serotonergic activity, which itself has been reported in numerous studies of violent individuals . In the present study, 25 violent psychiatric patients participated in a microbehavioral experience sampling procedure to examine differences in self-reports of affective and cognitive experiences as a function of serum cholesterol concentrations . For 7 days, they wore signaling devices that emitted an average of seven signals a day . Following each signal, patients filled out a mood questionnaire . Total serum cholesterol (TSC) concentration was positively associated with measures of affect, cognitive efficiency, activation, and sociability, suggesting a link between low TSC and dysphoria . These findings are consistent with the cholesterol-serotonin hypothesis and with the substantive literature linking both aggression and depression to depressed central serotonergic activity.

J Food Prot, 2001 Jan, 64(1), 94 - 8
Changes in K value and microorganisms of tilapia fillet during storage at high-pressure, normal temperature; Ko WC et al.; This study determined the effect of high-pressure, normal temperature (25 degrees C) storage on tilapia fillets . After pressurization, the fillets were stored at normal condition (1 atm {1 atm = 101.29 kPa} and 25 degrees C) for 12 h to evaluate the changes of microbes and K value . The fillets stored at 2,000 atm for 12 h still kept the K value, a freshness index that represents putrefaction with the value beyond 60%, still below 40%, and the K value of the meat without pressurization was up to 92% . Total plate counts of the fillets stored at below 1,000 atm for 12 h were maintained at the value of 4.7 log CFU/g of meat, which was similar to the initial level . However, the counts were obviously decreased to about 2.0 log CFU/g of meat for the fillets stored at above 2,000 atm . The same effect was obtained for psychrophilic bacteria . Enzymes and microbes reactivated apparently after 12 h of normal condition storage of mild pressurized fillets . The study demonstrates that high-pressure storage can inhibit the putrefaction of tilapia meat but no longer after that.

Electrophoresis, 2001 Jan, 22(1), 23 - 8
Hundredfold productivity of genome analysis by introduction of microtemperature-gradient gel electrophoresis; Biyani M et al.; Genome profiling, which employs temperature-gradient gel electrophoresis (TGGE) for DNA analysis, has recently been developed in identifying species by genotype . However, the performance of this technology like the general applications of TGGE was, though highly informative, limited in its ability due to methodological reasons . This study demonstrates that minimization of the gel for TGGE, to around one-tenth of its conventional size (approximately 2 cm), can be successfully introduced, resulting in a hundredfold higher performance (total evaluation of time, cost, and degree of parallel operations) than that of the conventional . Reproducibility was evaluated from the measures of the pattern similarity scores (PaSS) between band patterns (genome profiles) obtained with the conventional TGGE, and that with micro-TGGE (microTGGE) developed here, after extracting a set of featuring points from genome profiles . Size minimization, which leads to the reduction of the amount of samples required (cost-saving), is another great advantage, enhancing the employment of multicolor fluorescence technology . Since the further development of microbe-related fields such as epidemiology and microbial ecology inevitably require knowledge based on the identification of a great number of species and strains, microbe-related fields will receive the most optimal benefits from the technological improvements attained here.

Nature, 2001 Jan 11, 409(6817), 188 - 91
Nitrogen limitation of microbial decomposition in a grassland under elevated CO2; Hu S et al.; Carbon accumulation in the terrestrial biosphere could partially offset the effects of anthropogenic CO2 emissions on atmospheric CO2 . The net impact of increased CO2 on the carbon balance of terrestrial ecosystems is unclear, however, because elevated CO2 effects on carbon input to soils and plant use of water and nutrients often have contrasting effects on microbial processes . Here we show suppression of microbial decomposition in an annual grassland after continuous exposure to increased CO2 for five growing seasons . The increased CO2 enhanced plant nitrogen uptake, microbial biomass carbon, and available carbon for microbes . But it reduced available soil nitrogen, exacerbated nitrogen constraints on microbes, and reduced microbial respiration per unit biomass . These results indicate that increased CO2 can alter the interaction between plants and microbes in favour of plant utilization of nitrogen, thereby slowing microbial decomposition and increasing ecosystem carbon accumulation.

Anal Chem, 2001 Jan 1, 73(1), 1 - 7
Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays; Nelson BP et al.; Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species . In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array . For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected . The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity . Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides . Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface . DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).

Theriogenology, 2000 Nov 1, 54(8), 1281 - 4
Mummification of fetal membranes in the bovine vagina: a case report; Jalakas M; Papyraceous, mummified fetal membranes were found in the vagina of a cow at the end of the fourth month after a normal delivery . The uterus and cervix were without any pathological changes . The cow conceived on Day 36 after removal of the mummified fetal membranes upon the second insemination . Apart from the uncommon nature of the case, it proves that after calving the bovine vagina can maintain a sterile environment and that the constrictive mechanism of the vagina can be strong enough to hinder for a long time the discharge of fetal membranes and access of microbes to the vagina.

Cell Stress Chaperones, 2000 Nov, 5(5), 458 - 61
Regulation of immune activity by mild (fever-range) whole body hyperthermia: effects on epidermal Langerhans cells; Ostberg JR et al.; Inflammation of the skin and systemic fever, both of which occur with injury or infection, include a hyperthermic component that many believe constitutes a physiological stress . Such increases in local or systemic body temperature may also have a regulatory effect on immune function . Langerhans cells (LCs), the dendritic cells of the skin, continuously monitor the extracellular matrix of the skin by taking up particles and microbes that they then carry to draining lymph nodes for presentation to T lymphocytes . We hypothesize that the thermal element of inflammation and/or fever may help regulate the activation and migration of LCs out of the epidermis . To test this hypothesis, Balb/ c mice were exposed to a mild (39.8 degrees C +/- 0.2 degrees C), long-duration (6 hours) whole body hyperthermia (WBH) treatment, which mimics the thermal component of fever . The number of LCs and their morphology were analyzed at various time points up to 7 days after the initiation of WBH . The LCs of the ear epidermis were visualized using a fluorescein isothiocyanate-conjugated antibody specific for the major histocompatibility complex (MHC) class II molecule and confocal microscopy . Although MHC class II staining was diffuse on the surface of the LC body and dendritic extensions of both WBH and control samples, the WBH-treated LCs exhibited a more punctate morphology with fewer dendritic processes compared with control LCs . A significant decrease in the number of LCs was also observed 1 to 5 days after WBH treatment . Furthermore, in vitro heating of Balb/c ear skin cultures at 40 degrees C for 6 to 8 hours enhanced the numbers of viable LCs that migrated into the culture wells . These results suggest that WBH treatment stimulates epidermal LCs in the absence of foreign antigen.

Nippon Jibiinkoka Gakkai Kaiho, 2000 Nov, 103(11), 1218 - 26
{Isolation of FDC-lymphocyte clusters from human tonsillar tissues}; Takemoto N; Follicular B cells and follicular dendritic cells (FDCs) form FDC-lymphocyte clusters and play a central role in events related to humoral immunity in the lymphoid follicle (LF) . The secondary LF is divided into five zones, each of which exhibits functional differences . However, the distribution of the clusters across the five follicular zones remains unclear . We here report a procedure for isolating FDC-lymphocyte clusters from fixed tonsillar tissues and compare of the structure of clusters isolated from three follicular zones: the mantle, light and dark zones . First, the germinal centers (GCs) of the secondary LFs were removed under a stereoscope, and the GCs were enzymatically digested for 20, 30, 40 and 50 minutes at 37 degrees C . The FDC-lymphocyte clusters were then isolated using a discontinuous density gradient and a Magnetic Particle Concentrator, followed by microbeads . The number of isolated medium sized clusters composed of 6-25 cells was greatest when the samples were incubated for 40 minutes . To detect the mantle, light and dark zones, and GCs, isolated FDC-lymphocyte clusters from each zone were immunostained . Their cell structures were then compared . The clusters were composed mainly of B cells (comprising about 80% of the cells in each cluster, on average), T cells, natural killer/T cells and macrophages were also observed, but less frequently . The proportions of CD 45 RO-positive cells and CD4-positive cells were clearly different for each zone, with CD4-positive cells in the majority . No clear differences in isolated clusters from fixed and unfixed tonsillar tissues were observed . Our data indicate that this procedure is suitable for isolating FDC-lymphocyte clusters from fixed lymphoid tissues and that the proportions of cells composing the clusters differ in the three follicular zones.

Traffic, 2000 Apr, 1(4), 295 - 300
Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide; Sugita M et al.; A crucial feature of peptide antigen presentation by major histocompatibility complex (MHC) class I and II molecules is their differential ability to sample cytosolic and extracellular antigens . Intracellular viral infections and bacteria that are taken up in phagosomes, but then escape from the endocytic compartment efficiently, enter the class I pathway via the cytosol . In contrast, phagosome-resident bacteria yield protein antigens that are sampled deep in the endocytic compartment and presented in a vacuolar acidification-dependent pathway mediated by MHC class II molecules . Despite this potential for antigen sampling, microbes have evolved a variety of evasive mechanisms that affect peptide transport in the MHC class I pathway or blockade of endosomal acidification and inhibition of phagosome-lysosome fusion that may compromise the MHC class II pathway of antigen presentation . Thus, besides MHC class I and II, a third lineage of antigen-presenting molecules that bind lipid and glycolipid antigens rather than peptides exists and is mediated by the family of CD1 proteins . CD1 isoforms (CD1a, b, c, and d) differentially sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are differentially delivered . These CD1 pathways include vacuolar acidification-independent pathways for lipid antigen presentation . These features of presenting lipid antigens, independently monitoring various antigen-containing intracellular compartments and avoiding certain evasive techniques employed by microbes, enable CD1 molecules to provide distinct opportunities to function in host defense against the microbial world.

Plant J, 2001 Feb, 25(3), 281 - 93
The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface; Chiou TJ et al.; The movement of phosphate from the soil into plant root cells is the first of many crucial transport events required to supply phosphorous (P) to cells throughout the plant . In addition to the ability to acquire phosphate from the soil, the majority of the vascular plants are able to form arbuscular mycorrhizal associations in which phosphate may be delivered to the cortex via a fungus . Previously, we cloned two phosphate transporter genes, MtPT1 and MtPT2 from Medicago truncatula roots . Complementation of a yeast phosphate transport mutant revealed that MtPT1 is a functional phosphate transporter and Northern analyses revealed that MtPT1 is expressed exclusively in roots (Liu et al., 1998, Mol . Plant-Microbe Interact . 11, 14--22) . Utilising an antibody specific for MtPT1, we have analysed the accumulation and spatial expression patterns of the MtPT1 transporter . MtPT1 transcript and protein levels show close correlation and increase dramatically in the roots in response to phosphate starvation . MtPT1 protein levels decrease in roots during development of a symbiosis with arbuscular mycorrhizal (AM) fungi, indicating that this transporter is not involved in symbiotic phosphate transport . Membrane fractionation and analysis of a MtPT1/GFP fusion protein revealed that MtPT1 is located in the plasma membrane, while in situ hybridisation and immunolocalisation demonstrate the presence of MtPT1 transcripts and protein in the epidermal cells and root hairs of M . truncatula roots . MtPT1 shows expression patterns consistent with a role specifically in the acquisition of phosphate from the soil and is distinct from the other phosphate transporter of this class described to date.

Cell Microbiol, 1999 Nov, 1(3), 205 - 14
Phagocytic processing of antigens for presentation by class II major histocompatibility complex molecules; Ramachandra L et al.; Microbes and other particulate antigens (Ags) are internalized by phagocytosis and then reside in plasma membrane-derived phagosomes . The contribution of phagosomes to the degradation of Ags has long been appreciated . It has been unclear, however, whether peptides derived from these degraded antigens bind class II major histocompatibility complex (MHC-II) molecules within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes . Recent experiments have demonstrated that phagosomes containing Ag-conjugated latex beads express a full complement of Ag-processing molecules, e.g . MHC-II molecules, invariant chain, H2-DM and proteases sufficient to degrade bead- associated Ag . These phagosomes mediate the formation of peptide-MHC-II complexes, which are transported to the cell surface and presented to T cells . Phagosomes acquire both newly synthesized and plasma membrane-derived MHC-II molecules, but the formation of peptide-MHC-II complexes in phagosomes primarily involves newly synthesized MHC-II molecules . The content and traffic of phagosomal proteins vary considerably with the type of Ag ingested . Pathogenic microbes can alter phagosome composition and function to reduce Ag processing . For example, Mycobacterium tuberculosis blocks the maturation of phagosomes and reduces the ability of infected cells to present exogenous soluble protein Ags.

Radiat Res, 2001 Mar, 155(3), 402 - 8
The bystander effect in radiation oncogenesis: II . A quantitative model; Brenner DJ et al.; There is strong evidence that biological response to ionizing radiation has a contribution from unirradiated "bystander" cells that respond to signals emitted by irradiated cells . We discuss here an approach incorporating a radiobiological bystander response, superimposed on a direct response due to direct energy deposition in cell nuclei . A quantitative model based on this approach is described for alpha-particle-induced in vitro oncogenic transformation . The model postulates that the oncogenic bystander response is a binary "all or nothing" phenomenon in a small sensitive subpopulation of cells, and that cells from this sensitive subpopulation are also very sensitive to direct hits from alpha particles, generally resulting in a directly hit sensitive cell being inactivated . The model is applied to recent data on in vitro oncogenic transformation produced by broad-beam or microbeam alpha-particle irradiation . Two parameters are used in analyzing the data for transformation frequency . The analysis suggests that, at least for alpha-particle-induced oncogenic transformation, bystander effects are important only at small doses-here below about 0.2 Gy . At still lower doses, bystander effects may dominate the overall response, possibly leading to an underestimation of low-dose risks extrapolated from intermediate doses, where direct effects dominate.

Radiat Res, 2001 Mar, 155(3), 397 - 401
The bystander effect in radiation oncogenesis: I . Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells; Sawant SG et al.; It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation . Recently, evidence has accumulated that in cell populations exposed to low doses of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles . The end points observed include chromosome aberrations, mutations and gene expression . The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of alpha particles, and to assay for oncogenic transformation . The single-cell microbeam delivered no, one, two, four or eight alpha particles through the nuclei of all or just 10% of C3H 10T1/2 cells . We show that (a) more cells can be inactivated than were actually traversed by alpha particles and (b) when 10% of the cells on a dish are exposed to alpha particles, the resulting frequency of induced transformation is not less than that observed when every cell on the dish is exposed to the same number of alpha particles . These observations constitute evidence suggesting a bystander effect, i.e., that unirradiated cells are responding to damage induced in irradiated cells . This bystander effect in a biological system of relevance to carcinogenesis could have significant implications for risk estimation for low-dose radiation.

Toxicol Appl Pharmacol, 2001 Feb 15, 171(1), 61 - 9
Inflammatory responses in mice after intratracheal instillation of spores of Streptomyces californicus isolated from indoor air of a moldy building; Jussila J et al.; Microbial growth in buildings is associated with respiratory symptoms in the occupants . However, the specific effects of the microbes and the way they provoke clinical manifestations are poorly understood . In the current study, mice were exposed via intratracheal instillation to single doses of the spores of Streptomyces californicus, isolated from indoor air of a moisture-damaged building (2.2 x 10(7), 1.1 x 10(8), and 3.3 x 10(8) spores), or lipopolysaccharide (50 microg) . Inflammation and toxicity in lungs were evaluated 24 h later . The time course of the effects was explored with the dose of 1.1 x 10(8) spores for up to 7 days . The microbial spores elevated proinflammatory cytokine (i.e., TNFalpha and IL-6) levels in bronchoalveolar lavage fluid (BALF) and in serum in a dose- and time-dependent manner and evoked expression of inducible nitric oxide synthase in BAL cells . Both TNFalpha and IL-6 responses peaked at 6 h after instillation, but TNFalpha leveled off more quickly than IL-6 . The cytokine surge was followed by inflammatory cell recruitment into airways . Moreover, the spores increased dose- and time-dependently total protein, albumin, hemoglobin, and lactate dehydrogenase concentrations in BALF during the first 24 h . Histopathological examination of lungs confirmed the inflammatory changes . With the exception of macrophage and lymphocyte numbers, all parameters returned to control level at 7 days . In summary, these observations indicate that the spores of S . californicus are capable of provoking an acute inflammation in mouse lungs and can cause cytotoxicity . Thus, S . californicus can be considered as a species with potential to cause adverse health effects in occupants of moisture-damaged buildings.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1376 - 80
Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail; Schonbrunn E et al.; Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides . We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography . The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft . Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme.substrates complex . The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

J Hosp Infect, 2000 Dec, 46(4), 241 - 56
The index of microbial air contamination; Pasquarella C et al.; The standard index of microbial air contamination (IMA) for the measurement of microbial air contamination in environments at risk is described . The method quantifies the microbial flow directly related to the contamination of surfaces coming from microbes that reach critical points by falling on to them . The index of microbial air contamination is based on the count of the microbial fallout on to Petri dishes left open to the air according to the 1/1/1 scheme (for 1h, 1m from the floor, at least 1m away from walls or any obstacle) . Classes of contamination and maximum acceptable levels have been established . The index of microbial air contamination has been tested in many different places: in hospitals, in food industries, in art galleries, aboard the MIR space station and also in the open air . It has proved to be a reliable and useful tool for monitoring the microbial surface contamination settling from the air in any environment .

Cytometry, 2001 Feb 1, 43(2), 154 - 60
The "vanishing counting bead" phenomenon: effect on absolute CD34+ cell counting in phosphate-buffered saline-diluted leukapheresis samples; Brando B et al.; BACKGROUND: Using a single-platform protocol to count absolute CD34+ hematopoietic precursor cell (HPC) levels with different reference microbeads, we recorded occasionally artifactually high CD34+ HPC counts in some leukapheresis bags, whereas dual-platform calculations were always consistent . Abnormal countings were observed only when phosphate-buffered saline (PBS)-diluted leukapheresis samples were vortexed before analysis . A large series of blood samples analyzed similarly for CD34+ and CD4+ absolute counts did not show any sample or vortexing effect . With the volumetric absolute counting cytometer Partec-PAS, lower counts were also observed when different reference beads were vortexed before the instrument checking procedures . The counting abnormality was caused by a drop in microbead concentration (the "vanishing bead phenomenon") . This phenomenon reduced the total and relative bead event number in experimental and routine samples and in calibration procedures . This altered the bead denominator used to calculate absolute CD34+ HPC levels and it also reduced the concentration of standard calibration beads . METHODS: Using the Partec-PAS to measure volumetrically the actual bead concentration, we studied the vanishing bead phenomenon . Different types of counting and reference microbeads were resuspended in media with or without proteins or cells . Replicates were submitted either to gentle manual mixing or to vortexing before counting . RESULTS: Vortex agitation almost invariably induced the vanishing bead phenomenon when beads were resuspended in saline media or when an insufficient protein concentration was present, such as in diluted leukapheresis samples . Different bead types showed various degrees of sensitivity to vortexing . The bead disappearance was not caused by bubble formation or disruption . The addition of small amounts of protein completely prevented the vanishing bead phenomenon . The causative effect of the electrostatic charging of tube induced by vortexing is hypothesized . CONCLUSIONS: Sample suspensions containing counting beads for single-platform analysis must be resuspended in media with protein supplements to prevent the vanishing bead phenomenon and to ensure accurate counting .

Immunology, 2001 Jan, 102(1), 2 - 7
Co-option of endocytic functions of cellular caveolae by pathogens; Shin JS et al.; It is increasingly becoming clear that various immune cells are infected by the very pathogens that they are supposed to attack . Although many mechanisms for microbial entry exist, it appears that a common route of entry shared by certain bacteria, viruses and parasites involves cellular lipid-rich microdomains sometimes called caveolae . These cellular entities, which are characterized by their preferential accumulation of glycosylphosphatidylinositol (GPI)-anchored molecules, cholesterol and various glycolipids, and a distinct protein (caveolin), are present in many effector cells of the immune system including neutrophils, macrophages, mast cells and dendritic cells . These structures have an innate capacity to endocytoze various ligands and traffic them to different intracellular sites and sometimes, back to the extracellular cell surface . Because caveolae do not typically fuse with lysosomes, the ligands borne by caveolar vesicles are essentially intact, which is in marked contrast to ligands endocytozed via the classical endosome-lysosome pathway . A number of microbes or their exotoxins co-opt the unique features of caveolae to enter and traffic, without any apparent loss of viability and function, to different sites within immune and other host cells . In spite of their wide disparity in size and other structural attributes, we predict that a common feature among caveolae-utilizing pathogens and toxins is that their cognate receptor(s) are localized within plasmalemmal caveolae of the host cell.

Immunol Cell Biol, 2001 Feb, 79(1), 62 - 6
Genetic 'budget' of viruses and the cost to the infected host: a theory on the relationship between the genetic capacity of viruses, immune evasion, persistence and disease; Chaston TB et al.; The nature of the pathogen-host relationship is recognized as being a dynamic coevolutionary process where the immune system has required ongoing adaptation and improvement to combat infection . Under survival pressure from sophisticated immune responses, adaptive processes for microbes, including viruses, have manifested as immune evasion strategies . This paper proposes a theory that virus immune evasion can be broadly classified into 'acquisition' or 'erroneous replication' strategies . Acquisition strategies are characteristic of large genome dsDNA viruses, which (i) replicate in the cell nucleus; (ii) have acquired host genes that can be used to directly manipulate responses to infection; (iii) are often latent for the lifetime of the host; and (iv) have little or no serious impact on health . Alternatively, erroneous replication strategies are characteristic of small genome RNA viruses, which are recognized as being the cause of many serious diseases in humans . It is proposed that this propensity for disease is due to the cytoplasmic site of replication and truncated temporal relationship with the host, which has limited or removed the evolutionary opportunity for RNA viruses to have acquired host genes . This has resulted in RNA viruses relying on error-prone replication strategies which, while allowing survival and persistence, are more likely to lead to disease due to the lack of direct viral control over potentially host-deleterious inflammatory and immune responses to infection.

Plant Sci, 2000 Dec 7, 160(1), 1 - 13
Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils; Lopez-Bucio J et al.; During the last 20 years increasing experimental evidence has associated organic acid metabolism with plant tolerance to environmental stress . Current knowledge shows that organic acids not only act as intermediates in carbon metabolism but also as key components in mechanisms that some plants use to cope with nutrient deficiencies, metal tolerance and plant-microbe interactions operating at the root-soil interphase . In this review we summarize recent knowledge on the physiology and occurrence of organic acids in plants and their special relevance concerning nitrate reduction, phosphorus and iron acquisition, aluminum tolerance and soil ecology . We also discuss novel findings in relation to the biotechnological manipulation of organic acids in transgenic models ranging from cell cultures to whole plants . This novel perspective of organic acid metabolism and its potential manipulation may represent a way to understand fundamental aspects of plant physiology and lead to new strategies to obtain crop varieties better adapted to environmental and mineral stress.

Cancer Lett, 2001 Jan, 162 Suppl, S11 - S16
Predictive laboratory diagnostics in oncology utilizing blood-borne cancer cells--current best practice and unmet needs; Brandt BH et al.; The aim of laboratory diagnostics in oncology is to improve the clinical outcome of cancer by allowing earlier detection . Molecular knowledge of cancer should increase the number of risk and prognostic factors and will allow development of methods for detection and elimination of even very small tumors . Thus, the race for the specific tumor antigen in peripheral blood and the race for the blood-borne cancer cell happened simultaneously . The direct detection of the cells which have the highest probability to harbor all the properties mandatory to be life-threatening, conceivably metastatic, would be the most promising way to find the target structure of malignancy . Methods applying enrichment techniques based on density, morphology, tissue specific protein and tumor-associated protein detection enabled multi-parametric analysis of those blood-borne cancer cells . In exemplary studies it was demonstrated that the count of cell clusters positive for the tissue-specific proteins cytokeratin and prostate-specific antigen (PSA) from the peripheral blood of prostate cancer patients and a combination of a tissue-specific protein, a oncogenic receptor protein cytokeratin and p185(c-erbB-2) from the peripheral blood of breast cancer patients is related to the stage of the diseases . Breast cancer patients who presented with cytokeratin/p185(c-erbB-2) positive cell clusters showed a decrease of those cells under adriamycin adjuvant therapy . Nevertheless, additional molecular markers are required to characterize the functional properties of blood-borne cancer cells . Therefore, the genome of the cells can be investigated using a procedure for indirectly detecting aberrations of defined gene locations, i.e . multiplex microsatellite polymerase chain reaction . Up to now, the methods applied to the separation of blood-borne cancer cells are time-consuming and rather expensive . They consist of an initial enrichment step of density gradient centrifugation or buffy coat preparation followed by a specific isolation step using superparamagnetic microbeads coupled to antibodies, filter techniques or multi-parametric flow cytometry . Novel technologies have to be applied using miniaturization, integration and parallel-processing techniques based on those used in the computer industry to overcome the drawbacks.

J Clin Virol, 2001 Jan, 20(1-2), 1 - 6
Automation of laboratory testing for infectious diseases using the polymerase chain reaction-- our past, our present, our future; Jungkind D; While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process . Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor system, PCR has become an increasingly useful and widespread clinical tool . However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory . The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades . The automated Roche COBAS AMPLICOR system has automated the amplification and detection process . Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays . A new automated specimen preparation system, the COBAS AmpliPrep, was evaluated . The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin-streptavidin binding reaction . Once attached to the beads, the target is purified and concentrated automatically . Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively . The AmpliPrep instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample . This was a decrease of 76% compared to the manual specimen preparation method . Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future.

Aquat Toxicol, 2001 Mar, 52(1), 61 - 71
The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium; Cheng SH et al.; Embryonic vascular patterns in zebrafish (Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads . This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos . Zebrafish embryos were incubated in culture medium containing 100 microM cadmium from 5 h post fertilisation (hpf) to 48 hpf . At 48 hpf, embryos were examined for viability and occurrence of malformations . The 100 microM cadmium caused 32.21 +/- 3.65% mortality and 20.33 +/- 4.04% visible malformations in surviving embryos . In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed . Assessments on craniofacial development were made by digital measurements on areas of brains and eyes . Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain . Body lengths of the embryos were also measured . Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls . Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 microm into the circulation . All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature . Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions . The number of branch points in the vascular network was counted . In untreated embryos, there were 135.6 +/- 51 branches in the vasculature in entire body . In the cadmium treated embryos, there were 64.5+/-31 branches . The difference was significant when assessed with Student's t-test . It appeared that although cadmium did not cause any signs of external malformations in these visibly healthy embryos, nonetheless induced impaired branching and anastomsis of the cranial vessels . This study revealed, for the first time, that vital vascular structures in fish embryos could be affected by exposure to cadmium . This technique allowed visualisation of vascular anomalies in embryos showing no external signs of malformations . The impairment of anatomical features during embryonic development might serve as meaningful health endpoints in ecotoxicological studies and in risk assessment.

Environ Res, 2001 Feb, 85(2), 135 - 44
Exposure assessment of indoor allergens, endotoxin, and airborne fungi for homes in southern Taiwan; Su HJ et al.; This study was undertaken to examine the seasonal variations of domestic Der p 1, Der p 2, and endotoxin on mattress and airborne fungal concentrations in homes of asthmatic and nonasthmatic children in southern Taiwan, where temperature and relative humidity are usually high throughout the year . A group of asthmatic children (10-12 years old) were selected randomly based on a citywide questionnaire survey . The nonasthmatic children were chosen to be in the comparison group by matching in age, gender, and proximity of residence . Environmental sampling of domestic microbes was conducted once a month for a year . Twelve calendar months were grouped into spring, summer, fall, and winter according to weather data (mainly average temperature and humidity) from the Central Weather Bureau . Dust samples from a child's mattress and airborne samples from a child's bedroom were collected and analyzed for allergens of Der p 1 and Der p 2, endotoxin, and fungi respectively . Results show that about 65% of children's mattresses in our region have Der p 1 levels greater than 2 microg/g . It is also apparent that most airborne fungal concentrations found in homes of either asthmatic or nonasthmatic children are higher than the recommended levels of concern . The predominant genera are Cladosporium, Aspergillus, Penicillium, Alternaria, and yeast . In addition, seasonal effects seem to be a critical factor for the concentrations and distributions of domestic endotoxin in these study homes . The implication of long-term exposure to these high levels of environmental microbes and how their effects vary with seasons remain to be further characterized .

Plant Physiol, 2001 Feb, 125(2), 728 - 37
LEPS2, a phosphorus starvation-induced novel acid phosphatase from tomato; Baldwin JC et al.; Phosphate (Pi) is one of the least available plant nutrients found in the soil . A significant amount of phosphate is bound in organic forms in the rhizosphere . Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants . In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase . LePS2 is a member of a small gene family in tomato . The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide . The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken . Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases . Many plant homologs of LePS2 are found in the databases . The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi . However, the induction is repressible in the presence of Pi . Divided root studies indicate that internal Pi levels regulate the expression of LePS2 . The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses . The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate . The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase.

Am J Clin Nutr, 2001 Feb, 73(2 Suppl), 393S - 398S
Quality assurance criteria for probiotic bacteria; Tuomola E et al.; Acid and bile stability and intestinal mucosal adhesion properties are among the criteria used to select probiotic microbes . The quality control of probiotic cultures in foods traditionally has relied solely on tests to ensure that an adequate number of viable bacteria are present in the products throughout their shelf lives . Viability is an important factor, but not the only criterion for quality assurance . To be effective, probiotic strains must retain the functional health characteristics for which they were originally selected . Such characteristics include the ability to survive transit through the stomach and small intestine and to colonize the human gastrointestinal tract . In vitro test protocols can be readily adopted to examine the maintenance of a strain's ability to tolerate acidic conditions, survive and grow in the presence of bile, and metabolize selective substrates . Molecular techniques are also available to examine strain stability . Adhesion characterization may be an important quality-control method for assessing gut barrier effects . Adhesion has been related to shortening the duration of diarrhea, immunogenic effects, competitive exclusion, and other health effects . Adhesion properties should be carefully monitored, including adhesion to intestinal cells (eg, Caco-2) and human intestinal mucus . This article outlines the types of in vitro testing that can be used to ensure quality control of functional probiotic strains.

J Immunol, 2000 Apr 15, 164(8), 3946 - 9
Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox; Banerjee R et al.; The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line . In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-) . However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA . RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells . Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo . Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.

Int J Radiat Biol, 1999 Aug, 75(8), 1015 - 9
Lethality of single-track events: comparison between calculations and experimental data; Sato Y et al.; Recent results on cell killing by microbeams were analysed using three parameters (k, L, L1), where k is the mean number of lethal particles per cell nucleus, L {keV/microm} is the track-average LET (linear energy transfer) in cells, and L1 {keV/microm} is a critical value for inducing lethal damage by a single track . Analysis showed that calculations are consistent with two data sets . The existence of a quadratic dependence on LET of cellular effects is confirmed in the high-LET region between 30 and 500 keV/microm . L1 approximately 150 keV/microm was found to give the best fit and the sensitive area of the cell nucleus was determined as approximately 50 microm2 for Chinese hamster V79 cells . In microbeam experiments with several MeV alpha-particles, the relationship between k and L for V79 cells can be expressed as L = approximately 150(k)-1/2 . For a given survival level, the difference in the required dose (or L) between the microbeam and broad-beam experiments is also analytically described.

Transplantation, 2000 Dec 27, 70(12), 1752 - 60
Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses; Mathew JM et al.; BACKGROUND: The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established . METHODS: Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells . RESULTS: Compared with spleen cells as controls, un-irradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports) . The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34+ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages . These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38+, CD2+, CD5+, and CD1+ lymphoid cells (all were depleted of CD3+ cells) as well as CD33+ (but CD15 negative) myeloid precursors . However, positively selected CD19+ B-cells and CD15+ myeloid cells did not inhibit the MLR and CML responses . The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001) . Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3+ TcR-alphabeta+ CD4+ and CD28+ cells . CONCLUSION: A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro . Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.

FASEB J, 2001 Jan, 15(1), 59 - 69
Surfactant protein A (SP-A): the alveolus and beyond; Khubchandani KR et al.; Surfactant protein A (SP-A) is the major protein component of pulmonary surfactant, a material secreted by the alveolar type II cell that reduces surface tension at the alveolar air-liquid interface . The function of SP-A in the alveolus is to facilitate the surface tension-lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on surfactant function . It has also been shown that SP-A modulates host response to microbes and particulates at the level of the alveolus . More recently, several investigators have reported that pulmonary surfactant phospholipids and SP-A are present in nonalveolar pulmonary sites as well as in other organs of the body . We describe the structure and possible functions of alveolar SP-A as well as the sites of extra-alveolar SP-A expression and the possible functions of SP-A in these sites.

Ann Biomed Eng, 2000, 28(10), 1184 - 93
A chamber to permit invasive manipulation of adherent cells in laminar flow with minimal disturbance of the flow field; Levitan I et al.; An obstacle to real-time in vitro measurements of endothelial cell responses to hemodynamic forces is the inaccessibility of the cells to instruments of measurement and manipulation . We have designed a parallel plate laminar flow chamber that permits access to adherent cells during exposure to flow . The "minimally invasive flow device" (MIF device) has longitudinal slits (1 mm wide) cut in the top plate of the chamber to allow insertion of a recording, measurement, or stimulating instrument (e.g., micropipette) into the flow field . Surface tension forces at the slit openings are sufficient to counteract the hydrostatic pressure generated in the chamber and thus prevent overflow . The invasive probe is brought near to the cell surface, makes direct contact with the cell membrane, or enters the cell . The slits provide access to a large number (and choice) of cells . The MIF device can maintain physiological levels of shear stress (<1-15 dyn/cm2) without overflow in the absence and presence of fine instruments such as micropipettes used in electrophysiology, membrane aspiration, and microinjection . Microbead trajectory profiles demonstrated negligible deviations in laminar flow near the surface of target cells in the presence of microscale instruments . Patch-clamp electrophysiological recordings of flow-induced changes in membrane potential were demonstrated . The MIF device offers numerous possibilities to investigate real-time endothelial responses to well-defined flow conditions in vitro including electrophysiology, cell surface mechanical probing, local controlled chemical release, biosensing, microinjection, and amperometric techniques.

J Indian Med Assoc, 2000 Jul, 98(7), 377 - 80
Evolution of drug resistance and interpretation of drug sensitivity test; Pal D; Though with the advent of more and more numbers of antibiotics, infection control has become easier but it has been established more resistant strains of microbes are appearing . These resistant strains are becoming major public health problem . Drug misuse is one of the factors for resistant strains . While discussing mechanisms of drug resistance, gene transfer plays an important part . Drug resistance can be prevented by taking several strategies . There are several methods for performing antibiotic sensitivity test e.g., diffusion methods and dilution methods which are discussed elaborately.

Mem Inst Oswaldo Cruz, 2000, 95 Suppl 1, 153 - 8
Fungal infections in the immunocompromised host; Wanke B et al.; In recent years many remarkable changes occurred in our way of life, producing opportunities for microbes . All these changes are related to the recent emergence of previously unrecognized diseases, or the resurgence of diseases that, at least in developed countries, were thought to be under control . This concept is reviewed regarding fungal infections and their agents in the immunocompromised host . The changing pattern of these infections, the portals of entry of fungi into the human host, fungal pathogenicity and the main predisposing factors are analyzed . Opportunistic fungal infections in cancer, organ transplant and acquired immunodeficiency syndrome patients are reviewed, specially candidiasis and aspergillosis.

Vaccine, 2000 Dec 8, 19(9-10), 1038 - 46
Respiratory syncytial virus infection of gene gun vaccinated mice induces Th2-driven pulmonary eosinophilia even in the absence of sensitisation to the fusion (F) or attachment (G) protein; Bembridge GP et al.; Complete protection against respiratory syncytial virus (RSV) infection was induced in mice vaccinated on two occasions with 2.5 microg of DNA, encoding the fusion (F) protein of RSV, precipitated onto gold microbeads . In contrast, immunisation with DNA encoding the attachment (G) protein of RSV resulted in a significant reduction in viral load following infection, but did not afford complete protection . Gene gun delivery of DNA-F elicited a T helper-2 (Th2) biased immune response that could not be modulated by the co-delivery of plasmids encoding IL-2, IL-12 or IFNgamma . Similarly gene gun delivery of DNA-G primed a Th2 response . Thus, all gene gun vaccinated mice produced a predominant Th2 biased pulmonary immune response characterised by the production of IL-4 and IL-5 with little IFNgamma following RSV challenge . Analysis of bronchoalveolar lavage (BAL) cells, 5 days post challenge, indicated that there was only a two-fold increase in the number of inflammatory cells in vaccinated compared with control animals . Despite the strong Th2 cytokine bias of lung lymphocytes and the predominant recruitment of CD4(+) T cells, following challenge, there was not a marked pulmonary eosinophilic response (range from 2 to 7% of BAL) . In contrast, the BAL from mice vaccinated with control plasmid contained significantly more eosinophils than any other group.

Indian J Pediatr, 1999, 66(1 Suppl), S124 - 34
Diarrhoea and malnutrition interaction; Patwari AK; Epidemiological studies have demonstrated a marked negative relationship between diarrhoea and physical growth and development of a child . Each day of illness due to diarrhoea produces a weight deficit of 20-40 gms . Poor nutrition is associated with more serious prolonged diarrhoea . 'Catch-up growth' often does not occur in malnourished children . Malnutrition, particularly wasting, is a strong predictor of diarrhoeal duration and the prolonged illness could exacerbate nutritional faltering, thereby increasing the subsequent risk of death . Poor appetite, vomiting, deliberate withholding of food resulting in poor intake; malabsorption of macro and micronutrients; hastening of intestinal transit time; disturbance of metabolic and endocrine functions; and direct loss of protein and other nutrients in gastrointestinal tract are some of the known mechanisms which have an impact on the nutrition during an episode of diarrhea . In addition diarrhoea of infectious origin causes cytokine induced malnutrition which results from the actions of proinflammatory cytokines like tumour necrosis factor and interleukin 1, 6 and 8 . Preexisting malnutrition is associated with decreased turnover of epithelial cells resulting in delayed recovery which may prolong an episode of infectious diarrhoea by itself as well as by promoting tissue invasion by other enteropathogens . Malnutrition may also alter protective host factors and thereby favour intestinal colonization by the pathogenic microbes . Mucosal damage varying from moderately severe changes to flat lesions indistinguishable from those of celiac disease may occur in kwashiorkar . Diarrhoea malnutrition interaction represents a dangerous web which can be distangled by prevention of disease transmission by promoting exclusive breast feeding, hygienic weaning practices, safe drinking water and handwashing, improved host defences by breast feeding, improved nutrition, measles vaccine and other vaccines against enteropathogens in the offing; and promotion of standard case management with special emphasis on nutritional support and rehabilitation.

J Biol Chem, 2001 Apr 6, 276(14), 11100 - 12 Epub 2000 Dec 15.
Cuticular pro-phenoloxidase of the silkworm, Bombyx mori . Purification and demonstration of its transport from hemolymph; Asano T et al.; Pro-phenoloxidase (proPO) in insects is implicated in the defense against microbes and wounding . The presence of proPO in the cuticle was suggested more than 30 years ago, but it has not been purified . The extract of cuticles of the silkworm, Bombyx mori, was shown to contain two proPO isoforms (F-type and S-type proPOs, which have slightly different mobilities in polyacrylamide gel electrophoresis under nondenaturing conditions) . The two isoforms were purified to homogeneity . From hemolymph of the same insect, two types of proPO with the same electrophoretic mobilities as those of cuticular isoforms were separated and were shown to be different at five amino acid residues in one of their subunits . The isoforms in the hemolymph and cuticle were activated by a specific activating enzyme . The resulting active phenoloxidases exhibited almost the same substrate specificities and specific activities toward o-diphenols . The substrate specificities and the susceptibilities to inhibitors, including carbon monoxide, indicated that the purified proPO isoforms were not zymogens of laccase-type phenoloxidase . The proPO in hemolymph was shown to be transported to the cuticle . This demonstration was corroborated by the failure to detect proPO transcripts by Northern analysis of total RNA from epidermal cells . In reversed-phase column chromatography, cuticular and hemolymph proPOs gave distinct elution profiles, indicating that some yet to be identified modification occurs in hemolymph proPO and results in the formation of cuticular proPO . There was little transportation of cuticular proPO to the cuticle when it was injected into the hemocoel . The nature of the modification is described in the accompanying paper (Asano, T., and Ashida, M . (2001) J . Biol . Chem . 276, 11113-11125).

AANA J, 2000 Jun, 68(3), 233 - 6
Laryngoscope handles: a potential for infection; Simmons SA; Laryngoscope handles do not usually come in direct contact with the patient's mucous membranes . Consequently, routine disinfection of laryngoscope handles is not currently standard practice unless gross contamination is clearly evident . Recent reports indicate that apparently clean handles may be contaminated with blood or body fluids . No report examined microbes on handles . The present article describes the incidence and types of microbes on laryngoscope handles after their use in the operating rooms of a 502-bed medical center in northwestern Pennsylvania . Twenty laryngoscope handles were cultured on Mueller Hinton 5% sheep blood agar plates . The plates were incubated at 37 degrees C for 48 hours and examined for growth . The identification, incidence, and susceptibility patterns of organisms were determined . Microorganisms were present on all 20 laryngoscope handles . Nine different types were isolated; some strains were resistant to multiple antibiotics . Organisms were categorized as contaminants or opportunistic pathogens . The presence of opportunistic pathogens places anesthesia providers and patients at risk of nosocomial infections . Based on the recommendations of the 1997 American Association of Nurse Anesthetists' Infection Control Guide and the results of the present study, institutional guidelines should be established for the use of disposable laryngoscope covers, high-level (destroying all microorganisms with the exception of high numbers of bacterial spores) disinfection, or sterilization of laryngoscope equipment between each patient use.

Cancer J, 2000 Nov-Dec, 6(6), 343 - 50
Radiation, the two-edged sword: cancer risks at high and low doses; Hall EJ; Diagnostic Radiology and Radiation Oncology represent beneficial uses of radiation . The possible price tag is that radiation can cause, as well as diagnose or cure, cancer . Cancer risks at high doses are well known from epidemiologic studies of the Japanese survivors . Risks at low doses must be extrapolated from the high-dose data . The standard-setting bodies recommend a linear no-threshold extrapolation, but this is controversial . A knowledge of mechanisms, while not replacing epidemiology as a source of radiation-induced cancer risks, may provide insights into the shape of the dose-response relationships, and therefore on the validity of the linear extrapolation . The spectrum of second malignancies in radiotherapy patients sheds some light on mechanisms . More useful are new experiments involving the single-particle microbeam; these have demonstrated a bystander effect, that is, biologic effects in cells that are not directly irradiated, as well as mutations in cells where the cytoplasm, but not the nucleus, has been traversed by an alpha-particle.

Pediatr Clin North Am, 2000 Dec, 47(6), 1197 - 209
Immune function; Fleisher TA et al.; The innate and the adaptive immune systems have evolved to provide a rapid and specific means for protecting hosts against the many microbes experienced over a lifetime . These two immune responses interact cooperatively to enhance the host defense . Defects in either of these two pathways can have devastating consequences, as evidenced {figure: see text} by primary immune deficiencies, many of which are discussed in this issue of the Pediatric Clinics of North America . The immune system has a central role in the pathogenesis of many disorders that involve an inflammatory response, including allergic and autoimmune diseases . New and more effective therapies for these many disorders will develop as the understanding of the immune system and its many secreted mediators continues to increase.

Semin Thromb Hemost, 2000, 26(5), 589 - 94
The hepatic microvascular responses to sepsis; Ring A et al.; The liver is believed to play a major role in the initiation of multiorgan failure, the most lethal complication in the clinical course of sepsis . Microbes and their virulence factors enter the hepatic circulation where they first activate sinusoidal endothelial cells and Kupffer cells to produce proinflammatory mediators, including TNF-alpha, IL-1, IL-6, reactive oxygen metabolites, and eicosanoids . These mediators cause not only microbial killing, but also structural and functional liver damage concerning mainly the parenchymal cells . Leukocytes are targeted to the liver sinusoids by chemoattractants and, like platelets, tether to the sinusoidal endothelial cells, which are in a procoagulant state of inflammatory activation . Clogging of the sinusoids by these cells leads to a decrease of blood flow through the sinusoids, which is further aggravated by endothelin-1 effectuating the constriction of hepatic stellate cells in the sinusoids . In contrast, both nitric oxide (NO) and carbon monoxide (CO) act as antagonists of endothelin-1 by mediating relaxation of sinusoidal vessels . By maintaining an adequate sinusoidal perfusion, both NO and CO are hepatoprotective during the early, hyperdynamic phase of sepsis characterized by an increased cardiac output and moderate peripheral vasodilation . However, during the late, hypodynamic phase of sepsis, massive overproduction of NO by the inducible NO synthase leads to circulatory collapse, which inevitably includes breakdown of the liver circulation.

Haematologia (Budap), 2000, 30(3), 149 - 57
A solid phase and microtiter plate hemagglutination method for pretransfusion compatibility testing; Sandler SG et al.; Most hospital blood transfusion services perform routine pretransfusion compatibility tests (ABO/D typings, antibody detection tests and crossmatches) using standard test tube methods . Recently, alternative technologies, including microtiter plate methods, solid phase red cell adherence (SPRCA) assays, gel tests, microbead columns and affinity column assays have become available . While the increased sensitivity of these new serological technologies is an important advantage, cost savings and automated testing are also important benefits . Our hospital's Transfusion Service converted from manual test tube methods for compatibility testing to manual microtiter plate and SPRCA methods and, subsequently, to automated microtiter plate and SPRCA methods . The conversion was facilitated by using commercially-marketed reagent kits and a fully-automated blood typing analyzer . The automated blood typing system was linked electronically to a hand-held combination bar code reader/portable data terminal that enabled positive identification of patients' bar code wrist bands, personal identification badges, and bar code labels on patients' blood samples and blood components . This bar code identification system has been implemented in the hospital's outpatient Infusion Service . Thus, the conversion to microtiter plate and SPRCA assays enhanced transfusion safety not only by increasing the sensitivity of serological testing, but also by standardizing compatibility testing, supporting electronic record keeping, and linking the laboratory analyzer to a bar code identification system.

Am J Physiol Gastrointest Liver Physiol, 2001 Jan, 280(1), G1 - 6
Microbes and microbial toxins: paradigms for microbial-mucosal interactions I . Pathophysiological aspects of enteric infections with the lumen-dwelling protozoan pathogen Giardia lamblia; Eckmann L et al.; Giardia lamblia is one of the most important causes of waterborne diarrheal disease worldwide, and giardiasis is the most common protozoan infection of the human small intestine . Symptomatic infection is characterized by diarrhea, abdominal pain, and malabsorption, leading to malnutrition and weight loss, particularly in children . The pathogen resides strictly in the lumen of the small intestine, and infection is typically not accompanied by significant mucosal inflammation . Clinical and experimental studies indicate that B cell-dependent host defenses, particularly IgA, are important for controlling and clearing Giardia infection, although B cell-independent mechanisms also contribute to this outcome . In contrast to antigiardial host defenses, much less is known about the pathophysiological mechanisms underlying the clinical symptoms of giardiasis, partly because of the current lack of suitable model systems . In addition to being an important human enteric pathogen, Giardia is an interesting model organism for gaining basic insights into genetic innovations that led to evolution of eukaryotic cells, since it belongs to the earliest diverging eukaryotic lineage known . The completion of the giardial genome project will increase understanding of the basic biology of the protozoan and will help us to better understand host pathogen-interactions as a basis for developing new vaccination and therapeutic strategies.

Insect Mol Biol, 2000 Dec, 9(6), 661 - 73
Wolbachia infections in native and introduced populations of fire ants (Solenopsis spp.); Shoemaker DD et al.; Wolbachia are cytoplasmically inherited bacteria that induce a variety of effects with fitness consequences on host arthropods, including cytoplasmic incompatibility, parthenogenesis, male-killing and feminization . We report here the presence of Wolbachia in native South American populations of the fire ant Solenopsis invicta, but the apparent absence of the bacteria in introduced populations of this pest species in the USA . The Wolbachia strains in native S . invicta are of two divergent types (A and B), and the frequency of infection varies dramatically between geographical regions and social forms of this host . Survey data reveal that Wolbachia also are found in other native fire ant species within the Solenopsis saevissima species complex from South America, including S . richteri . This latter species also has been introduced in the USA, where it lacks Wolbachia . Sequence data reveal complete phylogenetic concordance between mtDNA haplotype in S . invicta and Wolbachia infection type (A or B) . In addition, the mtDNA and associated group A Wolbachia strain in S . invicta are more closely related to the mtDNA and Wolbachia strain found in S . richteri than they are to the mtDNA and associated group B Wolbachia in S . invicta . These data are consistent with historical introgression of S . richteri cytoplasmic elements into S . invicta populations, resulting in enhanced infection and mtDNA polymorphisms in S . invicta . Wolbachia may have significant fitness effects on these hosts (either directly or by cytoplasmic incompatibility) and therefore these microbes potentially could be used in biological control programmes to suppress introduced fire ant populations.

Radiat Res, 2001 Jan, 155(1 Pt 1), 122 - 6
Long-term genomic instability in human lymphocytes induced by single-particle irradiation; Kadhim MA et al.; Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations . Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that can be delivered . In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing alpha particles, that of a single particle traversal . Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single alpha-particle traversals on the surviving progeny of cells . A significant increase in the proportion of aberrant cells is observed 12-13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype . These data suggest that instability may be important in situations where even a single particle traverses human cells.

Radiat Res, 2001 Jan, 155(1 Pt 1), 89 - 94
Microdosimetry of a 25 keV electron microbeam; Wilson WE et al.; Electron microbeam experiments are planned or under way to explore in part the question regarding whether the bystander effect is a general phenomenon or is restricted to high-LET radiation . Since low-LET radiations scatter more readily compared to high-LET radiations, identifying bystander cells and assessing the potential dose that they may receive will be crucial to the interpretation of radiobiological results . This paper reports on initial calculations of the basic information needed for a stochastic model of the penetration of energetic electrons in tissue-like matter; the model will be used to predict doses delivered to adjacent regions in which bystander cells may reside . Results are presented of calculations of the stochastics of energy deposition by 25 keV electrons slowing down in a homogeneous water medium . Energy deposition distributions were scored for 1-micrometer spheres located at various penetration and radial distances up to 10 micrometer from the point of origin . The energy of 25 keV was selected because experiments are planned for that energy . At 25 keV there is a high probability that the entire electron track will be contained within a typical mammalian cell . Individual tracks are scored because of their primacy; data for higher doses can be obtained by convoluting single-track distributions . The event frequency decreases approximately exponentially after the first micrometer to 1% at about 8 micrometer of penetration . Radially, the 1% contour extends to 3.5 micrometer at a penetration of 5.5 micrometer . The frequency-mean energy deposited decreases from 1.5 to 1 keV/micrometer at a penetration of 3.5 micrometer, then increases back to about 1.5 at a penetration of 6.5 micrometer . The mean energy increases to about 3 keV/micrometer at a radial distance of 8.5 micrometer.

Infect Immun, 2001 Jan, 69(1), 494 - 500
Microtubule- and dynein-mediated movement of Orientia tsutsugamushi to the microtubule organizing center; Kim SW et al.; The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm . Orientia tsutsugamushi, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol . ECV304 cells infected with O . tsutsugamushi revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay . Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs . By transfection study (overexpression of dynamitin {also called p50}, which is known to associate with dynein-dependent movement), the movement of O . tsutsugamushi to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor . Although the significance of this movement in the life cycle of O . tsutsugamushi was not proven, we propose that the cytosolic O . tsutsugamushi bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.

Arch Environ Contam Toxicol, 2001 Jan, 40(1), 10 - 7
The effect of mercury and PCBs on organisms from lower trophic levels of a Georgia salt marsh; Wall VD et al.; We examined several indicators of salt marsh function, focusing on primary producers, microbes, and grass shrimp, at a Superfund site (LCP) contaminated with mercury and polychlorinated biphenyls (PCBs) and a reference site (Cross-River) in Georgia . Primary production of Spartina alterniflora was assessed by measuring peroxidase activity (POD), glutathione concentration (tGSH), photosynthesis (A(net)), and transpiration (E) . Microbial populations were assessed by measuring living-fungal standing crop (as ergosterol) and Microtox(R) . Grass shrimp (Palaemonetes pugio) reproductive potential was determined by measuring individual egg mass, average egg area, brood size, and brood mass of gravid females . Comparison of the sites suggested that P . pugio reproduction was affected at the LCP site, but we were unable to document clear negative effects on other organisms we investigated . Due to natural environmental gradients, the Cross-River site may not have been a perfect control for the LCP site . Therefore, data from just the LCP site were reanalyzed using multiple regression . Fungal biomass was related to methylmercury concentrations, but the direction of the relationship differed between wholly dead shoots (positive) and partially dead shoots (negative) . S . alterniflora POD was positively related to methylmercury concentrations . S . alterniflora A(net) and E were negatively related to elevation and salinity, respectively . Despite high levels of contamination at the LCP site, our results provided only suggestive evidence for impacts on organisms at lower trophic levels.

J Bacteriol, 2001 Jan, 183(1), 131 - 8
Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide; Spek EJ et al.; Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity . In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli . The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined . E . coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage {alkA tag strain}, oxidative damage {fpg nei nth strain}, and deaminated cytosine {ung strain}) showed essentially wild-type levels of NO(.) resistance . However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.) . In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing . Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.) . Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends . One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate . Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO( . ) can induce . Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.

Microbes Infect, 2000 Nov, 2(13), 1609 - 18
Leukocyte-facilitated entry of intracellular pathogens into the central nervous system; Drevets DA et al.; Microbes use numerous strategies to invade the central nervous system . Leukocyte-facilitated entry is one such mechanism whereby intracellular pathogens establish infection by taking advantage of leukocyte trafficking to the central nervous system . Key components of this process include peripheral infection and activation of leukocytes, activation of cerebral endothelial cells with or without concomitant infection, and trafficking of infected leukocytes to and through the blood-brain or blood-cerebrospinal fluid barrier.

Kekkaku, 2000 Oct, 75(10), 599 - 602
{Prospect of chemotherapy in the 21st century}; Omura S; "Golden Era in Chemotherapy" has begun with the discovery of penicillin in the early 1940's and lasted for two decades during which many antibiotics were discovered . However, the once-believed bright prospect that every infectious disease could be eliminated on the earth by the discovery of antibiotics had to be canceled owing to the emerging of drug-resistant microbes . It was indeed a rat race . We are now at the point when we have to seek another way to combat infectious diseases: One possible way might be not to eradicate the microbes but to coexist with them so long as they do no harm to the human hosts . The first step of infection with pathogens to the host is the adherence of the microbes to the surface of host cells . Therefore, the method how to inhibit this adhesion of microbes to the host cells may provide a new tool to prevent the development of infectious diseases without elimination of microbes from the host . This is just an example of strategy by which humans and pathogens coexist at peace and should be taken into consideration for the development of new-type antibiotics or "anti-infective drugs" in the 21st century . The analysis of genome sequences has been accelerated recently for various pathogenic bacteria one by one . New targets in the pathogenic microbes for the development of new antibiotics can, therefore, be determined from the genetic point of view . The discovery of antibiotics has indeed been the history of collection of innumerable species and/or strains of bacteria from the soils to search for the biologically active anti-pathogenic agents . The current progress in the technology of molecular genetics, however, will certainly make it possible to search for active molecules by DNA technology; bacterial DNA but not whole microorganisms from the soil is to be transformed into the conventional bacteria and searched for active molecules with combat against pathogens.

Kekkaku, 2000 Oct, 75(10), 595 - 8
{Immunology in the 20th century--progress made in research on infectious and immunological diseases}; Kishimoto T; The new era of the modern medicine was opened 100 years ago by Robert Koch and Louis Pasteur who demonstrated that various infectious diseases were caused by their respective microbes . Koch discovered Mycobacterium tuberculosis, the causative agent of tuberculosis . The first breakthrough in the modern medicine to combat against infectious diseases was the discovery of anti-diphtheria toxin antibody by E.A . von Behring and S . Kitasato . The concept of immunity--immune from disease--has thus been established . The immune response between antigen and antibody sometimes provides the host with a harmful effect . The concept of allergy was introduced by Richet and later by Prausnitz and Kustner . Why the same immune response leads to the different outcome, immunity or allergy had not been made clear until the discovery of IgE by Drs . Kimishige and Teruko Ishizaka in 1968: The IgG antibody plays a role in immunity whereas IgE antibody is involved in allergy . Tuberculin skin reaction which is well known as the diagnostic tool for mycobacterial infection was studied by M . Chase in 1945 demonstrating that it was able to be transferred to the healthy individual by immune cells but not by antibody . The immune response is now categorized into two; soluble immunity--immediate type allergy and cell-mediated immunity--delayed type allergy . The rapid progress in the molecular biology in the past decades has also accelerated the progress in immunology, several of which include discovery of two types of lymphocytes; T and B cells; concept of two T cells; Th1 and Th2 cells; and the discovery of cytokines which regulate immune cell responses . The mechanism of the immune response is now understood at the gene level . Several immunological diseases can now be successfully treated by controlling the levels of cytokines involved . For example, refractory rheumatoid arthritis is now under control by the administration of recombinant soluble TNF receptor molecules to the patients . The complete human genome sequence is currently under investigation . We can now envisage the advent of the days when every disease can be diagnosed and intervened at the gene level.

Int Microbiol, 1999 Mar, 2(1), 39 - 42
Snow algae of the Sierra Nevada, Spain, and High Atlas mountains of Morocco; Duval B et al.; Snow algae (Chlorophyta) are reported from the Sierra Nevada mountains in southern Spain and the High Atlas mountains of Morocco . Populations of the snow algae Chlamydomonas sp., coloring the snow orange-red, were collected from Pico de Veleta, Spain, while snow samples from Mt . Neltner in the High Atlas mountains, contained resting spores of an orange-green colored Chloromonas sp . Other microbes observed in snow samples include bacteria, fungi, heterotrophic euglenids, diatoms, nematodes, and heterotrophic mastigotes (flagellated protists) . This is the first report of snow algae from the Sierra Nevada mountains of Spain and from the Afro-alpine environment.

Int Microbiol, 1998 Dec, 1(4), 255 - 8
The renaissance of microbiology; Davies J; Microbiology is finally occupying its true position as the pre-eminent field in life sciences . This is due to advances in molecular techniques that confirm the evolutionary significance of the biology of microbes . It is anticipated that the use of comparative genomics will provide information that will advance the understanding of mechanisms of pathogenesis and the importance of secondary metabolism in social microbiology . More emphasis on studies of microbial diversity will increase its value in both fundamental microbiology and its industrial applications.

J Neurobiol, 2001 Jan, 46(1), 29 - 40
Contribution of neurons to habituation to mechanical stimulation in Caenorhabditis elegans; Kitamura KI et al.; In Caenorhabditis elegans, a light touch induces a locomotor response . Repeated touches, however, result in an attenuation of response, that is, habituation . Withdrawal responses elicited by anterior touch are controlled by anterior mechanosensory neurons (AVM and ALMs), and by four pairs of interneurons (AVA, AVB, AVD, and PVC) (Chalfie et al., 1985; White et al., 1986) . To identify the neurons that participate in habituation, we ablated these neurons with a laser microbeam and investigated the resulting habituation of the operated animals . The animals lacking both left and right homologues AVDLR were habituated more rapidly than intact animals . We propose that chemical synapses at AVD play a critical role in the habituation of intact animals .

PDA J Pharm Sci Technol, 2000 Nov, 54(6), 449 - 55
Development of a dye ingress method to assess container-closure integrity: correlation to microbial ingress
Burrell LS, Carver MW, DeMuth GE, Lambert WJ.
To demonstrate maintenance of parenteral product sterility, container-closure integrity over the shelf life of the product is critical . In the past, sterility testing has been used to ensure closure integrity . However, because of the limitations associated with sterility testing, there is a need for an improved method for evaluating container-closure integrity . This article describes the development of a physical test method (dye ingress) for the evaluation of container-closure integrity . FD&C Red No . 40 dye was used in dye ingress studies . The dye solution visual detection limit was similar to the spectophotometric detection limit . This limit was approximately 0.0025 microL of dye/mL, which corresponds to an absorbance of approximately 0.002 absorbance units at 506 nm . Breached vials with various sizes of microtubes were utilized to correlate the dye ingress method with a microbial ingress method . The inner diameter of the microtubes ranged from 2 to 75 microns . The dye ingress and microbial ingress methods had similar sensitivity to breached vials . One advantage of the dye method over microbial ingress is that it may be utilized with vials containing formulations that are cidal or static to microbes . Thus, the dye ingress method is considered an excellent test method for evaluating container-closure integrity.

Genome Biol . 2000;1(1):RESEARCH002 . Epub 2000 Apr 27.
Phylogenetic variation and polymorphism at the toll-like receptor 4 locus (TLR4); Smirnova I et al.; BACKGROUND: Differences in responses to bacterial surface lipopolysaccharides (LPSs) are apparent between and within mammalian species . It has been shown in mice that resistance to LPS is caused by defects in the Toll-like receptor 4 gene (Tlr4), the product of which is thought to bind LPS and mediate LPS signal transduction in immune system cells . RESULTS: We have sequenced the Toll-like receptor 4 gene of humans (TLR4; 19.0 kilobases, kb) and mice (Tlr4; 91.7 kb), as well as the coding region and splice junctions of Tlr4 from 35 mouse (Mus musculus) strains, from the chimpanzee and from the baboon . No other discernible genes or regions of interspecies conservation lies close to Tlr4 and, in both humans and mice, flanking sequences and introns are rich in repeats of retroviral origin . Interstrain analyses reveal that Tlr4 is a polymorphic protein and that the extracellular domain is far more variable than the cytoplasmic domain, both among strains and among species . The cytoplasmic domain of the Tlr4 protein is highly variable at the carboxy-terminal end . CONCLUSIONS: We suggest that selective evolutionary pressure exerted by microbes expressing structurally distinguishable LPS molecules has produced the high level of variability in the Tlr4 extracellular domain . The highly variable carboxy-terminal region of the cytoplasmic domain is likely to determine the magnitude of the response to LPS within a species.

J Dairy Sci, 2000 Nov, 83(11), 2580 - 4
Mixed ruminal microbes of cattle produce isopropanol in the presence of acetone but not 3-D-hydroxybutyrate; Bruss ML et al.; The objective was to evaluate the ability of mixed rumen microbes to synthesize isopropanol from acetone or 3-D-hydroxybutyrate . Rumen fluid from seven mature, nonpregnant, dry Holstein cows was incubated with starch or cellulose and additions of acetone, 3-D-hydroxybutyrate, or saline . Rumen fluid was analyzed for isopropanol after 0, 3, 6, and 9 h . No isopropanol was present in any sample at 0 h, and none was present in incubations containing saline or 3-D-hydroxybutyrate at any subsequent time . Incubations that included acetone produced small amounts of isopropanol from 0 to 3 and 3 to 6 h and significantly larger amounts from 6 to 9 h . With starch as the energy substrate, production from 6 to 9 h was 3.8 micromol/min per liter of rumen fluid and 3.7 micromol/min per liter with cellulose as the energy substrate; however, these values did not differ significantly . Mixed rumen microbes could synthesize isopropanol from acetone but not from 3-D-hydroxybutyrate, and rumen microbial metabolism of acetone was the likely source of plasma isopropanol seen in ketotic ruminants.

Br J Nutr, 2000 Oct, 84(4), 477 - 82
Portal recovery of short-chain fatty acids infused into the temporarily-isolated and washed reticulo-rumen of sheep; Kristensen NB et al.; The present study was undertaken to study the metabolism of short-chain fatty acids (SCFA) by the reticulo-ruminal epithelium and the portal-drained viscera (PDV) under in vivo conditions with no interference from the metabolism of the rumen microbes . The technique of temporary isolation of the reticulo-rumen was applied to wethers implanted with catheters in a mesenteric artery, the hepatic portal vein and the right ruminal vein . Portal blood flow was measured by downstream dilution of p-aminohippuric acid; the PDV uptake of arterial acetate, as well as the whole-body irreversible loss rate (ILR) of acetate, was estimated by {2-(13)C}acetate infusion into the right ruminal vein . The sheep were maintained with a bicarbonate-buffered solution of SCFA in the reticulo-rumen along with continuous intraruminal infusion of SCFA for 4 h . The portal appearance of SCFA of non-reticulo-ruminal origin was estimated before and after the infusion protocol . Of the acetate absorbed by the sheep, 89 (SE 5), 109 (SE 7) and 101 (SE 7)% was recovered as portal net appearance of acetate, portal net appearance of acetate corrected for PDV uptake of arterial acetate and increase in the ILR of acetate respectively . Of the propionate, isobutyrate, butyrate, isovalerate and valerate absorbed by the sheep, 95 (SE 7), 102 (SE 9), 23 (SE 3), 48 (SE 5) and 32 (SE 4)% respectively was recovered as portal net appearance . In contrast to current concepts, the present study showed that the reticulo-ruminal epithelium metabolizes none (or only a small proportion) of the acetate and propionate absorbed from the rumen . This observation could lead to the more efficient use of results obtained with multi-catheterized animals to quantify the net metabolite output of the rumen microbes.

Nature, 2000 Nov 23, 408(6811), 463 - 6
Variation in the reversibility of evolution; Teotonio H et al.; How reversible is adaptive evolution? Studies of microbes give mixed answers to this question . Reverse evolution has been little studied in sexual populations, even though the population genetics of sexual populations may be quite different . In the present study, 25 diverged replicated populations of Drosophila melanogaster are returned to a common ancestral environment for 50 generations . Here we show that reverse evolution back to the ancestral state occurs, but is not universal, instead depending on previous evolutionary history and the character studied . Hybrid populations showed no greater tendency to undergo successful reverse evolution, suggesting that insufficient genetic variation was not the factor limiting reverse evolution . Adaptive reverse evolution is a contingent process which occurs with only 50 generations of sexual reproduction.

Braz J Infect Dis, 1998 Apr, 2(2), 85 - 89
Percutaneous Injuries With Sharp Instruments and the Behavior of Anesthesiologists and Obstetricians in Regard to the Associated Risk of Occupational Infectious Diseases: A Survey in a Town in Brazil; de Andrade Noshioka S et al.; Health care workers are at risk of acquiring several infectious diseases, including human immunodeficiency virus (HIV) infection, from percutaneous injuries with contaminated needles and sharp instruments . A survey among anesthesiologists and obstetricians in a Brazilian town assessed their risk of these injuries and their behavior toward prophylaxis . Both anesthesiologists and obstetricians tended to be more adherent to universal precautions when caring for patients with known HIV positive status, although the difference was statistically significant only for the use of goggles . Of those surveyed, 80% of the obstetricians and 65% of anesthesiologists had at least 1 episode of injury with a needle or sharp instrument in the last 12 months . Injuries were mostly from syringe needles (88.9%) among the anesthesiologists, and from suture needles (56.9%) among the obstetricians . The annual risk of injury was much higher among obstetricians (2 per 100 procedures) than among anesthesiologists (11 per 10,000 anesthesias and 17 per 10,000 venous punctures/intravenous catheterizations) . The majority of the anesthesiologists and obstetricians referred for vaccination against hepatitis B received the vaccine as post-exposure prophylaxis . Promoting recognition of percutaneous injuries with sharp instruments among health care workers as an important occupational health problem, distribution of information on methods to prevent such injuries, and when to immunize are objectives yet to be achieved to prevent the transmission of infection by HIV and other microbes . Proper assessment of a number of new devices, techniques, and protective equipment may result in more effective preventive measures in the future.

Radiat Environ Biophys, 2000 Sep, 39(3), 173 - 7
Monte Carlo simulation of single-cell irradiation by an electron microbeam; Miller JH et al.; A model is presented for irradiation of a cellular monolayer by an electron microbeam . Results are presented for two possible window designs, cells plated on the vacuum-isolation window and cells plated on Mylar above the vacuum-isolation window . Even for the thicker dual-membrane window that facilitates tissue culture and allows the target cell to be centered relative to the electron beam, the majority of the calculated beam spreading was contained in a volume typical of the mammalian HeLa cell line . None of the 10(4) electrons simulated at 25 keV were scattered into the spatial region occupied by neighbors of the target cell . Dose leakage was largest at 50 keV where the mean energy deposited in all neighbors was 21% of that deposited in the target cell . This ratio was reduced to 5% at 90 keV, the highest beam energy simulated . Lineal energy spectra of energy deposition events scored in the nucleus of the target cell became progressively more like the gamma-ray spectrum as the electron beam energy increased . Hence, our simulations provide strong support for the feasibility of a low-LET, single-cell irradiator.

Recent Results Cancer Res, 2001, 158, 113 - 7
Morphologically intact melanoma cells may be detected in peripheral blood of melanoma patients; Benez A et al.; The detection of circulating melanoma cells has been the subject of numerous investigations in recent years . We developed a cellular approach to identifying circulating melanoma cells in peripheral blood using immunomagnetic cell sorting . The examination covered 205 blood samples from 155 melanoma patients and 30 samples from healthy persons and nonmelanoma patients . After density gradient centrifugation, the interphase was incubated with the 9.2.27 antibody . Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting . Cells were stained using an alkaline phosphatase-anti-alkaline phosphatase assay and examined by light microscopy . In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per milliliter of whole blood could be detected reliably . Circulating melanoma cells were not found in 30 controls, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma . In patients with regional lymph node metastases and disseminated disease, circulating 9.2.27-positive cells could be detected in 3 of 29 patients (10%) and 13 of 85 patients (15%) examined, respectively . We conclude that immunomagnetic cell sorting is a promising method with high sensitivity and specificity . The method is not suitable for early detection of metastases but is a valuable tool for further investigating the biological characteristics of circulating melanoma cells.

J Immunol, 2000 Dec 1, 165(11), 6107 - 15
Microbial lipopeptides induce the production of IL-17 in Th cells; Infante-Duarte C et al.; Naive Th cells can be directed in vitro to develop into Th1 or Th2 cells by IL-12 or IL-4, respectively . In vivo, chronic immune reactions lead to polarized Th cytokine patterns . We found earlier that Borrelia burgdorferi, the spirochaete that causes Lyme disease, induces Th1 development in alpha beta TCR-transgenic Th cells . Here, we used TCR-transgenic Th cells and oligonucleotide arrays to analyze the differences between Th1 cells induced by IL-12 vs those induced by B . burgdorferi . Transgenic Th cells primed with peptide in the presence of B . burgdorferi expressed several mRNAs, including the mRNA encoding IL-17, at significantly higher levels than Th cells primed with peptide and IL-12 . Cytometric single-cell analysis of Th cell cytokine production revealed that IL-17 cannot be categorized as either Th1 or Th2 cytokine . Instead, almost all IL-17-producing Th cells simultaneously produced TNF-alpha and most IL-17(+) Th cells also produced GM-CSF . This pattern was also observed in humans . Th cells from synovial fluid of patients with Lyme arthritis coexpressed IL-17 and TNF-alpha upon polyclonal stimulation . The induction of IL-17 production in Th cells is not restricted to B . burgdorferi . Priming of TCR-transgenic Th cells in the presence of mycobacterial lysates also induced IL-17/TNF-alpha coproduction . The physiological stimulus for IL-17 production was hitherto unknown . We show here for the first time that microbial stimuli induce the expression of IL-17 together with TNF-alpha in both murine and human T cells . Chronic IL-17 expression induced by microbes could be an important mediator of infection-induced immunopathology.

Sci Prog, 2000, 83 ( Pt 3), 223 - 42
The identification, examination and exploration of Antarctic subglacial lakes; Siegert MJ; At the floor of the Antarctic ice sheet, 4 km below the Russian research base Vostok Station, lies a 2,000 km3 body of water, comparable in size to Lake Ontario . This remote water mass, named Lake Vostok, is the world's largest subglacial lake by an order of magnitude (Figure 1) . Despite ice-surface temperatures regularly around -60 degrees C, the ice-sheet base is kept at the melting temperature by geothermal heating from the Earth's interior . The ice sheet above the lake has been in existence for at least several million years and possibly as long as 20 million years . The origins of Lake Vostok may therefore data back across geological time to the Miocene (7-26 Ma) . The hydrology of Lake Vostok can be characterised by subglacial melting across its northern side, and refreezing over the southern section . A deep ice core, located over the southern end of the lake has sampled the refrozen ice . Geochemical analysis of this ice has found that it comprises virtually pure water . However, normal glacier ice contains impurities such as debris and gas hydrates . Subglacial melting and freezing over Lake Vostok may, therefore, leave the lake enriched in potential nutrients issued from the melted glacier ice . Many scientists expect microbial life to exist within the lake, adapted to the extreme conditions of low nutrient and energy levels . Indeed microbes have been found in the basal refrozen layers of the ice sheet . If Lake Vostok has been isolated from the atmosphere for several million years by the ice sheet that lays above it, the microbes within the lake must also date back several million years and may have undergone evolution over this time, yielding life that may be unique to Lake Vostok . Plans are currently being arranged to explore Lake Vostok and other Antarctic subglacial lakes, and identify life in these extraordinary places . Before this happens, however, much more needs to be known about the ice-sheet above subglacial lakes, and the rocks and sediment below them.

Plant Sci, 2000 Nov 6, 159(2), 257 - 264
Variability in plant-microbe interaction between Lupinus lines and Bradyrhizobium strains; Robinson KO et al.; Even though lupin (Lupinus albus L.) is known to potentially fix 150-200 kg/ha nitrogen for the use of a succeeding crop, precise information about lupinxBradyrhizobium strain interaction under the climatic conditions prevalent in the mid-Atlantic region of the United States is unknown . We conducted two greenhouse experiments with the objective of characterizing this symbiotic relationship and to evaluate potential interaction between Bradyrhizobium strains and lupin lines . In the first experiment, performance of 60 bradyrhizobial strains was evaluated by inoculating three lupin cultivars and using combined score, which consisted of an arithmetic total of plant vigor, nodulation scores from crown root, nodulation scores from fibrous roots, shoot dry weight, and root dry weight . In the second experiment, performance of 80 lupin lines was evaluated by inoculating with three selected Bradyrhizobial strains and using the combined score, which consisted of an arithmetic total of plant vigor, acetylene reduction activity, nodule number per plant, nodule weight per plant, and dry shoot weight . Significant variation existed for all traits in both experiments except for nodule number in the second experiment . Significant Bradyrhizobial strain by lupin line interaction existed for nodulation score, shoot and root dry weights, and the combined scores . Comparison of relative ranks indicated that nodulation effectiveness was dependent on specific strain and lupin line combinations . It was concluded that specific Bradyrhizobial strain and lupin line combinations would need to be identified for successful utilization of lupin's capability to fix atmospheric nitrogen for use in low-input and sustainable agriculture.

ASDC J Dent Child, 2000 Sep-Oct, 67(5), 350 - 4, 302, 304
The effects of water filtration systems on fluoride: Washington, D.C . metropolitan area; Jobson MD et al.; According to the U.S . Environmental Protection Agency (EPA), approximately one in eight Americans is exposed to potentially harmful microbes, pesticides, lead, or radioactive radon whenever they drink a glass of tap water or take a shower . One reason for this exposure is that the water plants are aging or ill equipped to process the huge amounts of raw sewage and agricultural pollutants that are still being discharged into our drinking-water sources . Other compounds such as fluoride and chloride have been added to the community water supplies for health benefits . Water filtration systems are becoming more popular as people become concerned with pollutants in the public water supply and questions are being raised as to whether fluoride is affected by these filters . The aim of this pilot study was to assess the efficacy of three types of water filtration systems and to determine their impact on fluoride content of the water in the Washington, D.C . metropolitan area . One sample of water was collected daily for fourteen days, from one location . The sample was divided to use as a control and the test samples which were processed through various filter systems . With the use of a fluoride ion specific electrode, the fluoride concentration level was tested in all samples in order to determine the percentage of fluoride removed . This study was intended to prove that the water filtration systems did not affect the advantage offered by optimum water fluoride levels . The experimental samples were ascertained and compared to the control group, resulting in three of the four carbon filters showing statistically significant amounts of fluoride removed from the water . Both Reverse Osmosis and Distillation, as expected, removed the fluoride at a high rate.

Cardiovasc Surg, 2000 Dec, 8(7), 550 - 4
Procalcitonin (PCT) in cardiac surgery: diagnostic value in systemic inflammatory response syndrome (SIRS), sepsis and after heart transplantation (HTX); Boeken U et al.; PURPOSE: Since it is of great importance to distinguish between a systemic inflammatory response syndrome (SIRS) and an infection caused by microbes especially after heart transplantation (HTX), we examined patients following heart surgery by determining procalcitonin (PCT), because PCT is said to be secreted only in patients with microbial infections . METHODS: Sixty patients undergoing coronary artery bypass grafting (CABG) and 14 patients after heart transplantation were included in this prospective study . In the CABG group we had 30 patients without any postoperative complications (group A) . Furthermore we took samples of 30 patients who suffered postoperatively from a sepsis (group B, n=15) or a systemic inflammatory response syndrome (C, n=15) . In addition we measured the PCT-levels in 65 blood samples of 14 patients after heart transplantation (Group I: rejection > IIa, II: viral infection (CMV), III: bacterial/fungal infection, IV: controls) . RESULTS: In all patients of group A the pre- and intraoperative PCT-values and the measurement at arrival on intensive care unit (ICU) were less than 0.2 ng/ml . On the second postoperative day the PCT-value was 0.33+/-0.15 ng/ml in the control group . At the same time it was 19.6+/-6.2 ng/ml in sepsis and 0.7+/-0.4 ng/ml in systemic inflammatory response syndrome patients (P<0.05) . In transplanted patients we could find the following PCT-values: Gr.I: 0.18+/-0.06 II: 0.30+/-0.09 III: 1.63+/-1.16 IV: 0.21+/-0.09 ng/ml (P<0.05 comparing group III with I, II and IV) . CONCLUSIONS: These results show that extracorporeal circulation (ECC) and systemic inflammatory response syndrome do not initiate a PCT-secretion . Septic conditions cause a significant increase of PCT . In addition, PCT is a reliable indicator concerning the essential differentiation of bacterial or fungal--not viral--infection and rejection after heart transplantation.

Environ Exp Bot, 2000 Nov 1, 44(3), 207 - 219
Uptake capacity of amino acids by ten grasses and forbs in relation to soil acidity and nitrogen availability; Falkengren-Grerup U et al.; Uptake capacity of organic nitrogen was studied in solution experiments on eight grasses and two forbs growing in acid soils with relatively high nitrogen mineralisation in southern Sweden . Uptake of a mixture of amino acids (alanine, glutamine, glycine), that varied between 1.6 and 6.3 micromol g(-1) dw root h(-1), could not be explained by soil data from the species' field distributions (pH, total carbon and nitrogen, potential net mineralisation of ammonium and nitrate) . The ratio between organic and inorganic nitrogen (methylamine) uptake was <0.05 for the forbs, higher for the grasses with a maximum of 1.42 for Deschampsia flexuosa . The ratio was negatively correlated with measures related to soil acidity (Ellenberg's R-value, soil nitrate and total carbon) but not, as hypothesised, with the total amount of mineralised nitrogen . The total demand on nitrogen by all components of the ecosystem would probably have described the extent to which competition among and between plants and microbes induced nitrogen limitation . In a methodological study two grasses were exposed to pH 3.8, 4.5 and 6.0 and to 50, 100 and 250 micromol l(-1) of three amino acids . Uptake was also compared between intact plants and excised roots . The treatment response varied considerably between the species which stresses the importance of studying intact plants at field-relevant pH and concentrations.

Nat Biotechnol, 2000 Nov, 18(11), 1172 - 6
Removal of antibiotic resistance genes from transgenic tobacco plastids; Iamtham S et al.; Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes . Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally . Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin . We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants . A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes . The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich . Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene . Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.

Nat Immun, 1998, 16(5-6), 198 - 206
The concept of an immune mechanism of chemical homeostasis and its importance in biology and medicine; Bykova A et al.; This paper outlines research that has led to the concept of the inevitable participation of the immune system in an organism's chemical homeostasis . This function of the immune system is tentatively named the 'immune mechanism of the chemical homeostasis' (IMCH) . It is based on the theory of a permanent physiological synthesis of antibodies to endogenous biologically active substances . Minimal accumulation of biologically active substances as a result of the influence of different factors specifically activates the immune system in order to maintain its chemical homeostasis . The concept suggests the necessity of widening the notion of the range of the immune system's censorial functions . The concept explains the preexistence of immunocompetent cells preadapted to biologically active substances and autoantibodies specific to them; the natural clonality of the B lymphocyte pool; the polyclonal lymphocyte activation triggered by mitogens, foreign proteins, erythrocytes, and microbes, and tolerance to drugs.

Expert Opin Investig Drugs, 2000 May, 9(5), 975 - 91
Therapeutic potential of complement inhibitors in myocardial ischaemia; Lucchesi BR et al.; Under normal conditions, the complement system functions to eradicate microbes and other membrane bound pathogens . In other situations, complement activation comprises a pivotal mechanism for mediating tissue demolition in inflammatory disorders, including ischaemia/reperfusion injury . Complement-mediated tissue damage has long been recognised as a significant contributor to myocardial reperfusion injury . However, clinical use of complement inhibitors to reduce the extent of irreversible tissue injury related to reperfusion, remains in the early stages of development . Activation of the complement system generates anaphylatoxins, opsonins and the lytic moiety known as the membrane attack complex (MAC) . In addition, fragments of the complement cascade proteins (e.g., C3a and C5a) secondarily initiate processes deleterious to myocytes by recruiting and stimulating inflammatory cells, such as neutrophils and macrophages, within the area of reperfusion . Damaged tissue itself, is capable of upregulating the genes that encode the formation of complement proteins leading to assembly of the MAC, which in turn further advances tissue injury . All of these factors contribute to the development of myocardial infarction subsequent to ischaemia and reperfusion . This paper provides an overview of how the complement system operates and examines the various inhibitors, both endogenous and exogenous, that regulate the complement cascade . Activation and inhibition of the complement system will be discussed primarily in the context of myocardial ischaemia and reperfusion injury.

J Cell Sci, 2000 Nov, 113 ( Pt 22), 3979 - 87
Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells; Yan L et al.; The growth and regression of capillary blood vessels during angiogenesis is greatly influenced by changes in the activity of matrix metalloproteinases (MMPs), which selectively degrade extracellular matrix (ECM) and thereby modulate capillary endothelial cell shape, growth and viability . However, changes in cell-ECM binding and cell spreading have also been reported to alter MMP secretion and activation . Studies were carried out to determine whether changes in integrin binding or cell shape feed back to alter MMP-2 processing in human capillary endothelial (HCE) cells . Catalytic processing of proMMP-2 to active MMP-2 progressively decreased when HCE cells were cultured on dishes coated with increasing densities of fibronectin (FN), which promote both integrin binding and cell spreading . Conversely, the highest levels of active MMP-2 were detected in round cells cultured on low FN . When measured 24 hours after plating, this increase in active MMP-2 was accompanied by a concomitant rise in mRNA and protein levels for the membrane-type 1 MMP (MT1-MMP), which catalyzes the cleavage of proMMP-2 . To determine whether proMMP-2 processing was controlled directly by integrin binding or indirectly by associated changes in cell shape, round cells on low FN were allowed to bind to microbeads (4.5 microm diameter) coated with a synthetic RGD peptide or FN; these induce local integrin receptor clustering without altering cell shape . ProMMP-2 activation was significantly decreased within minutes after bead binding in these round cells, prior to any detectable changes in expression of MT1-MMP, whereas binding of beads coated with control ligands for other transmembrane receptors had no effect . This inhibitory effect was mimicked by microbeads coated with activating antibodies against alphaVbeta3 and beta1 integrins, suggesting a direct role for these cell-surface ECM receptors in modulating proMMP-2 activation . Similar inhibition of proMMP-2 processing by integrin binding, independent of cell spreading, was demonstrated in cells that were cultured on small, microfabricated adhesive islands that prevented cell spreading while presenting a high FN density directly beneath the cell . Interestingly, when spread cells were induced to round up from within by disrupting their actin cytoskeleton using cytochalasin D, proMMP-2 processing did not change at early times; however, increases in MT1-MMP mRNA levels and MMP-2 activation could be detected by 18 hours . Taken together, these results suggest the existence of two phases of MMP-2 regulation in HCE cells when they adhere to ECM: (1) a quick response, in which integrin clustering alone is sufficient to rapidly inhibit processing of proMMP-2 and (2) a slower response, in which subsequent cell spreading and changes in the actin cytoskeleton feed back to decrease expression of MT1-MMP mRNA and, thereby, further suppress cellular proteolytic activity.

Appl Environ Microbiol, 2000 Nov, 66(11), 4662 - 72
Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage; Buchan A et al.; Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the alpha-proteobacteria was investigated . Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the beta-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes . Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the beta-ketoadipate pathway . All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate) . Four of the Roseobacter group isolates had inducible protocatechuate 3, 4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate . The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity . Additional genes involved in the protocatechuate branch of the beta-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates . Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria . A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates . This evidence of protocatechuate 3, 4-dioxygenase and the beta-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

J Interferon Cytokine Res, 2000 Oct, 20(10), 843 - 55
Interleukin-1/Toll receptor family members: receptor structure and signal transduction pathways; Daun JM et al.; Interleukin-1 (IL-1) is a central mediator of the inflammatory response . It plays a role in both systemic and local immune responses to invading microbes . There are two receptors (IL-1RI and IL-1RII) that mediate the cellular responses . These receptors belong to a family of receptors based on homologous receptor structure within the intracellular signaling domain . Other family members include the Drosophila protein Toll, the recently discovered mammalian Toll-like receptors (TLR), and the IL-18 receptor . Engagement of these receptors by their diverse ligands results in activation of very similar signal transduction cascades through use of common signaling intermediates . These signal transduction cascades lead to the activation of cellular responses that are known to regulate the innate immune response . Therefore, elucidating the function and redundancy of this receptor family is essential to the understanding of the innate immune response . This review examines each member of this receptor family and emphasizes similarities and potential differences in both receptor structure and signal transduction pathways to further the understanding of this complex receptor family.

Am J Gastroenterol, 2000 Oct, 95(10), 2872 - 9
Shared and unique environmental factors determine the ecology of methanogens in humans and rats; Florin TH et al.; OBJECTIVE: This study ascertains the relative contributions of genetics and environment in determining methane emission in humans and rats . There is considerable interest in the factors determining the microbial species that inhabit the colon . Methanogens . which are archaebacteria, are an easily detected colonic luminal bacteria because they respire methane . They are present in some but not all human colons and lower animal hindguts . Opinion varies on the nature of the factors influencing this ecology with some studies proposing the existence of host genetic influences . METHODS: Methane emission was measured in human twin pairs by gas chromatography, and structural equation modeling was used to determine the proportion of genetic and environmental determinants . The importance of the timing of environmental effects and rat strain on the trait of methane emission were ascertained by experiments with cohabiting methanogenic and nonmethanogenic rats . RESULTS: Analysis of breath samples from 274 adolescent twin pairs and their families indicated that the major influences on the trait of methane emission are the result of shared (53%, 95% confidence interval 39-61) and unique environmental (47%, 95% confidence interval 38-56) effects . No significant autosomal genetic effects were detected, but as observed in other studies, men (37%) were less likely to excrete methane in their breath than women (63%) . Investigation of methane emission in rats indicated that environmental effects in this animal are most potent during the weaning period, with stable gut microbial ecology thereafter for some but not all rat strains . CONCLUSIONS: These results are consistent with shared and unique environmental factors being the main determinants of the ecology of this colonic microbe.

Curr Opin Microbiol, 2000 Oct, 3(5), 475 - 80
Assessing evolutionary relationships among microbes from whole-genome analysis; Eisen JA; The determination and analysis of complete genome sequences have recently enabled many major advances to be made in the area of microbial evolutionary biology . These include the determination of the first genome of a Crenarchaeota, the suggestion that horizontal gene transfer may be the rule rather than the exception, and revelations about how genomes evolve on short timescales.

Biol Trace Elem Res, 2000 Aug, 76(2), 161 - 73
Chemical forms of selenium present in rat and ram spermatozoa; Alabi NS et al.; In vivo and in vitro studies were conducted to investigate the chemical forms by ion-exchange chromatography of selenium (Se) present in rat and ovine spermatozoa . After injection with 75Se-selenite, the form of 75Se in rat sperm was selenocysteine, but selenocysteine and selenomethionine (SeMet) were present in ovine sperm . Presumably, synthesis of SeMet by rumen microbes are responsible for its presence in ovine sperm . In vitro incubation of ram sperm with selenocysteine or SeMet produced no changes, but incubation with selenite produced a compound that eluted one fraction before SeMet from the ion-exchange column . After treatment of this fraction with mercaptoethanol, it eluted in a later fraction upon rechromatography, suggesting it to be selenodicysteine . This compound is apparently formed because of high levels of cysteine in semen . Cysteine, reduced glutathione, and oxidized glutathione were also found in semen . The significance of the results is discussed.

Yale J Biol Med, 1999 Sep-Oct, 72(5), 321 - 8
Evolution of microbiology as seen in the textbooks of Edwin O . Jordan and William H . Park; Strick J; Historians of science account the appearance of textbooks as an important step in the formation and consolidation of a new discipline . The texts of Park and Jordan were both very important in this light; however; they also can be used as a gauge of changing concepts within microbiology in the first four decades after its consolidation as a discipline, 1900-1940 . This paper tracks these important texts and through them changing attitudes toward several important concepts: bacterial variation, human/bovine tuberculosis, and the existence of a non-symptomatic carrier state in infectious disease . The two texts are also compared regarding their view of microbes as pathogens vs . microbes as important and ubiquitous ecological agents.

J Dairy Sci, 2000 Oct, 83(10), 2326 - 34
Partitioning of amino acids flowing to the abomasum into feed, bacterial, protozoal, and endogenous fractions; Shabi Z et al.; We partitioned the flow of amino acids (AA) to the abomasum among rumen undegradable protein (RUP) and bacterial, protozoal, and endogenous fractions using four Holstein cows in midlactation that were equipped with ruminal and abomasal cannulas . A 2 x 2 factorial design with four diets, combinations of high or low ruminally degradable organic matter, and rumen degradable protein, was employed . Crude protein (CP) and AA contents of ruminal bacteria and protozoa and abomasal digesta were determined . Equations for the source compositions and in vivo flows of CP and 16 AA were then solved simultaneously with a linear program to estimate the contribution of RUP, bacterial, protozoal, and endogenous CP to AA flows . The flows of RUP and bacterial AA were not affected by diet . Low dietary RDP increased the flow of protozoal AA to the abomasum, but the ruminally degradable organic matter content of the diet did not affect protozoal AA flow . Across diets, RUP, bacterial, protozoal, and endogenous fractions provided 55, 33, 11, and <1% of the CP, and 62, 26, 12, and <1% of the AA that reached the abomasum . The linear program was a useful tool for partitioning AA that flows to the abomasum . The technique may also allow dietary effects on ruminal microbes and the AA profile of protein flowing to the duodenum to be better understood and perhaps manipulated.

J Chromatogr A, 2000 Sep 29, 893(1), 115 - 22
Effects of DNA topology, temperature and solvent viscosity on DNA retardation in slalom chromatography; Hirabayashi J et al.; Slalom chromatography is a unique size-fractionation method applicable to large DNA molecules {>5 kilobase pairs (kbp)} . The method was first developed by using columns packed with microbeads (diameter, <20 microm) used for high-performance liquid chromatography and by applying a relatively fast flow-rate (>0.3 ml/min) . Previous studies suggested that the separation is attributed to a hydrodynamic rather than to an equilibrium phenomenon (J . Hirabayashi and K . Kasai, Anal . Biochem . 178 (1989) 336; J . Hirabayashi, N . Itoh, K . Noguchi and K . Kasai, Biochemistry, 29 (1990) 9515) . In the present report, the results of a systematic study on the effects of DNA topology, temperature, and solvent viscosity on DNA retardation are described . Firstly, the behaviour of circular (super-coiled) and linearized forms of charomid DNAs (20-42 kbp) was studied . Circular-form DNA molecules were found to be fractionated size-dependently similarly to linear forms in a flow-rate dependent manner . However, the extent of retardation of the circular form DNA was apparently less than that of the corresponding linear forms . Circular DNAs showed almost the same retardation (e.g., 42 kbp) as DNA fragments (e.g., 20 kbp) having approximately half of the size of the former . This observation indicates that DNA retardation is basically related to physical length, not to mass . Secondly, to study the effect of temperature with special reference to solvent viscosity, we carried out chromatographic analysis at various temperatures ranging from 6 to 65 degrees C in both the absence and presence of sucrose (10 or 20%, w/v) . The results showed that it is the solvent viscosity that determines the extent of retardation . Taken together, all of physicochemical parameters that define hydrodynamic properties, i.e., particle size, flow-rate and solvent viscosity, proved to be critical in slalom chromatography as well as the potential physical length of the DNA, thus supporting the concept that slalom chromatography is based on a hydrodynamic principle.

J Plant Growth Regul, 2000 Jun, 19(2), 131 - 143
An Introduction to the Biosynthesis of Chemicals Used in Plant-Microbe Communication; Paiva NL; Plants accumulate a diverse array of natural products, which can serve either to defend the plant against various microbes in its environment or to attract various microbes, both beneficial and pathogenic . Plants must also attract pollinators, repel or poison herbivores, compete with other plant species, and protect themselves from environmental dangers such as high light intensities . Some compounds have been implicated in playing a role in multiple interactions . Although the structures vary immensely in size and complexity, most are derived from a limited number of core biosynthetic pathways . This review briefly summarizes the biosynthetic origins of phenylpropanoid (including simple phenolics, flavonoids, anthocyanins and isoflavonoids), polyacetate, terpenoid, and alkaloid classes of metabolites . Compounds reported to be important in plant-microbe, plant-animal, and plant-plant interactions will be given as examples of each of these classes . Other aspects of biosynthesis also will be discussed, including the timing or location of biosynthesis, the potential for genetic manipulation of these pathways, and various questions regarding the biosynthesis of these compounds.

J Immunol Methods, 2000 Oct 20, 244(1-2), 205 - 15
Isolation of endothelial cells from murine tissue; Marelli-Berg FM et al.; The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task . In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues . Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin . The single cell suspension obtained is then incubated with an anti-CD31 antibody, anti-CD105 antibody and with biotinylated isolectin B-4 . Pure EC populations are finally obtained by magnetic bead separation using rat anti-mouse Ig- and streptavidin-conjugated microbeads . EC cultures are subsequently expanded and characterised . The surface molecule expression by the primary cultures of murine EC obtained from lung and heart tissue is analysed and compared to that of a murine endothelioma and of primary cultures of murine renal tubular epithelial cells . The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage.

Shock, 2000 Sep, 14(3), 361 - 5
Hepatocyte toll-like receptor 2 expression in vivo and in vitro: role of cytokines in induction of rat TLR2 gene expression by lipopolysaccharide; Liu S et al.; We and others have demonstrated previously that cytokines, including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNFalpha), regulate LPS recognition proteins such as CD14 in the liver and on hepatocytes . Based on recent findings that the mammalian homologue of Drosophila Toll participates in LPS signaling, we examined the regulation of Toll-Like Receptor (TLR) gene expression by cytokines in vitro and its distribution in vivo with a focus on the liver as a site of host-microbe interaction . Our results show that IL-1beta and/or TNFalpha participate in the upregulation of TLR2 mRNA levels in hepatocytes . Rats treated concurrently with LPS and antagonists of the IL-1 or TNFalpha receptor demonstrated significantly reduced LPS-induced hepatic expression of TLR2 compared to animals treated with LPS alone . The increase in hepatic TLR2 mRNA expression was associated with enhanced transcription as determined by nuclear run-on analysis . LPS treatment in vivo caused a marked TLR2 mRNA up-regulation in all of the tissues examined, with liver showing the highest expression . The high level of TLR2 expression in the liver may have important implications for pathogen-host interactions or microbial signaling.

Biochem Biophys Res Commun, 2000 Oct 14, 277(1), 93 - 9
Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer; Alenghat FJ et al.; A magnetic tweezer was constructed to apply controlled tensional forces (10 pN to greater than 1 nN) to transmembrane receptors via bound ligand-coated microbeadswhile optically measuring lateral bead displacements within individual cells . Use of this system with wild-type F9 embryonic carcinoma cells and cells from a vinculin knockout mouse F9 Vin (-/-) revealed much larger differences in the stiffness of the transmembrane integrin linkages to the cytoskeleton than previously reported using related techniques that measured average mechanical properties of large cell populations . The mechanical properties measured varied widely among cells, exhibiting an approximately log-normal distribution . The median lateral bead displacement was 2-fold larger in F9 Vin (-/-) cells compared to wild-type cells whereas the arithmetic mean displacement only increased by 37% . We conclude that vinculin serves a greater mechanical role in cells than previously reported and that this magnetic tweezer device may be useful for probing the molecular basis of cell mechanics within single cells .

Sante, 2000 May-Jun, 10(3), 211 - 9
{Immunosuppression and vaccinations}; Debat Zoguereh D et al.; Immunocompromised subjects have a higher risk of infection . Some infectious risks could be controlled by vaccination, carried out according to normal schedules, in the absence of effective curative treatment . According to the type of immunodeficiency and its severity, certain vaccines may be recommended, others should be avoided and still others may be used with no particular risk . Thus, in immuno-compromised subjects, vaccines consisting of inactivated, inert or dead microorganisms are indicated . In contrast, those consisting of live microbes are not recommended for several reasons: 1) there is a risk that the immunocompromised host will be unable to control infection with the vaccine; 2) there is a risk of an increase in viral replication, particularly in HIV-infected subjects and 3) there is a risk that the response of the immune system will be poor . HIV-infected individuals now account for most of the cases of secondary immuno-deficiency, following the emergence of AIDS in 1980 . They are increasing in number, especially in developing countries, where antiretroviral treatment is not widely available . In this context, vaccinations against transmissible childhood viral and bacterial diseases (e.g . measles and polio) with a high prevalence is advisable, with modifications according to the risk of contagion and the degree of immunodeficiency . However, these vaccines do not target opportunist infections, the prognosis of which is poor and against which we have no vaccines . Other vaccines are recommended only for immunocompromised subjects visiting countries with specific epidemiological situations (endemic or epidemic diseases) or if vaccination is required by the country visited.

Biochim Biophys Acta, 2000 Jul 31, 1467(1), 85 - 90
Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells; Millot C et al.; We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro . After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis . Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.

Microbiology, 2000 Oct, 146 ( Pt 10), 2435 - 45
Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor; Joyner DC et al.; Ferric iron is an essential element for microbial growth but its water solubility in aerobic environments is considered to be low . Thus it is a limiting resource for which microbes must compete in natural habitats . Since competition for iron occurs at the level of individual cells, knowledge of the variability in iron bioavailability to such individuals is required to assess the nature of the competition in these habitats . Ferric iron availability to cells of Pseudomonas syringae was assessed by quantifying the fluorescence intensity of single cells harbouring a plasmid-borne transcriptional fusion of an iron-regulated promoter from a locus encoding a membrane receptor for a pyoverdine siderophore with a reporter gene encoding green fluorescent protein (GFP) following fluorescence microscopy . Cells of this iron biosensor exhibited iron-dependent GFP fluorescence that was inversely proportional to the amount of iron added to the media, and which differed by over 20-fold in iron-replete compared to iron-deplete culture media . Cells cultured in a medium of a given iron content exhibited a very narrow range of fluorescence intensities . In contrast, the fluorescence intensity of cells of the biosensor strain recovered from the rhizosphere or phylloplane of inoculated bean plants varied greatly . The distribution of fluorescence intensities was strongly right-hand skewed, with about 10% of the cells exhibiting substantially higher GFP fluorescence than that of the median cell . Cells of a positive control strain, harbouring a fusion of the constitutive nptII promoter with the gfp reporter gene, exhibited uniform GFP fluorescence both in culture media and on plants . These results indicate that there is substantial heterogeneity of iron biovailability to cells of P . syringae on plants, with only a small subset of cells experiencing low iron availability . Such heterogeneity places constraints on models of interactions of bacteria in natural habitats that are based on competition for limited iron.

Biochim Biophys Acta, 2000 Sep 27, 1487(2-3), 275 - 85
GDP-fucose: beta-galactoside alpha1,2-fucosyltransferase, MFUT-II, and not MFUT-I or -III, is induced in a restricted region of the digestive tract of germ-free mice by host-microbe interactions and cycloheximide; Lin B et al.; A shift from sialylation to fucosylation of mucosal glycoconjugates occurred in the mammalian digestive tract in the weaning period, but mice under germ-free conditions were found to express both fucosyl GM1 (FGM1) and fucosyl asialo GM1 (FGA1) in the stomach, cecum and colon, but not in the small intestine . By host-microbe interactions and administration of cycloheximide, FGA1 was quickly induced in the small intestine, but the concentrations of fucosylated glycolipids in the other regions were not altered significantly . Their expression coincided with the activity of GDP-fucose:GA1 alpha(1, 2)-fucosyltransferase (alpha1,2-FT), and we isolated a cDNA with an open reading frame encoding the murine alpha1,2-FT (MFUT-II) of 347 amino acids with a predicted molecular mass of 39.21 kDa . The intraperitoneal injection of cycloheximide induced the mRNA and activity of alpha1,2-FT (MFUT-II) in the small intestine of germ-free mice, whereas no change in the mRNA or activity was observed in the stomach, cecum and colon, indicating that expression of FGA1 in response to microbial colonization or cycloheximide is transcriptionally regulated in a restricted region of the murine digestive tract . At 24 h after the administration of cycloheximide, FGA1 was preferentially produced in the upper half of the duodenal microvilli.

Nat Med, 2000 Oct, 6(10), 1154 - 9
Dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can initiate antigen-specific humoral immunity independent of CD4+ T cells; Kikuchi T et al.; We have investigated whether dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can trigger B cells to produce antigen-specific antibodies without CD4+ T-cell help . Dendritic cells modified with a recombinant adenovirus vector to express CD40 ligand and pulsed with heat-killed Pseudomonas induced naive B cells to produce antibodies against Pseudomonas in the absence of CD4+ T cells in vitro, initiated Pseudomonas-specific humoral immune responses in vivo in wild-type and CD4-/- mice, and protected immunized wild-type and CD4-/-, but not B-cell -/- mice, from lethal intrapulmonary challenge with Pseudomonas . Thus, genetic modification of dendritic cells with CD40 ligand enables them to present a complex mixture of microbial antigens and establish CD4+ T cell-independent, B cell-mediated protective immunity against a specific microbe.

Cell Calcium, 2000 Jun, 27(6), 353 - 62
Dynamic ca(2+)changes in neutrophil phagosomes A source for intracellular ca(2+)during phagolysosome formation?
Lundqvist-Gustafsson H, Gustafsson M, Dahlgren C.
An increase in cytosolic Ca(2+)concentration periphagosomally is critical for phagolysosomal formation and neutrophil elimination of microbes . The Ca(2+)increase could be achieved through release of Ca(2+)from mobilized intracellular stores . Alternatively, Ca(2+)that passively enter the phagosome during phagocytosis could be provided by the phagosome . Intraphagosomal Ca(2+)changes in single human neutrophils was measured during phagocytosis of serum opsonized Fura-2-conjugated zymosan particles, using a digital image processing system for microspectrofluorometry . A decrease in phagosomal Ca(2+)down to nanomolar concentrations was seen within minutes following phagosomal closure . Blockage of plasma membrane Ca(2+)channels by econazole abolished this decrease . The fluorescence properties of Fura-2 zymosan were retained after phagocytosis and stable to pH changes, reactive oxygen species, and proteolytic enzymes . We suggest that Ca(2+)ions present in the phagosome enter the cell cytosol through Ca(2+)channels in the phagosomal membrane, achieving a localized Ca(2+)rise that is important for phagosome processing .

Phys Med Biol, 2000 Sep, 45(9), 2497 - 508
Monte Carlo simulation of dose distributions from a synchrotron-produced microplanar beam array using the EGS4 code system; Orion I et al.; Microbeam therapy is established as a general concept for brain tumour treatment . A synchrotron based x-ray source was chosen for experimental research into microbeam therapy, and therefore new simulations were essential for investigating the therapy parameters with a proper description of the synchrotron radiation characteristics . To design therapy parameters for tumour treatments, the newly upgraded LSCAT (Low energy SCATtering) package of the EGS4 Monte Carlo simulation code was adapted to develop an accurate self-written user code for calculating microbeam radiation dose profiles with a precision of 1 microm . LSCAT is highly suited to this purpose due to its ability to simulate low-energy x-ray transport with detailed photon interactions (including bound electron incoherent scattering functions, and linear polarized coherent scattering) . The properties of the synchrotron x-ray microbeam, including its polarization, source spectrum and beam penumbra, were simulated by the new user codes . Two concentric spheres, an inner sphere, defined as a brain, and a surrounding sphere, defined as a skull, represented the phantom . The microbeam simulation was tested using a 3 x 3 cm array beam for small treatment areas and a 6 x 6 cm array for larger ones, with different therapy parameters, such as beam width and spacing . The results showed that the microbeam array retained an adequate peak-to-valley ratio, of five times at least, at tissue depths suitable for radiation therapy . Dose measurements taken at 1 microm resolution with an 'edge-on' MOSFET validated the basics of the user code for microplanar radiation therapy.

Microbes Infect, 2000 Aug, 2(10), 1193 - 205
Bartonella henselae, B . quintana, and B . bacilliformis: historical pathogens of emerging significance; Karem KL et al.; Bartonella species were virtually unrecognized as modern pathogens of humans until the last decade . However, identification of Bartonella species as the agents of cat-scratch disease, bacillary angiomatosis, urban trench fever, and possible novel presentations of Carrion's disease has left little doubt of the emerging medical importance of this genus of organisms . The three primary human pathogenic bartonellae, Bartonella bacilliformis (Carrion's disease), B . henselae (cat-scratch disease), and B . quintana (trench fever), present noteworthy comparisons in the epidemiology, natural history, pathology, and host-microbe interaction that this review will briefly explore.

J Appl Physiol, 2000 Oct, 89(4), 1619 - 32
Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz; Maksym GN et al.; We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli . We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field . Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency . The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being approximately 0 . 35 at all frequencies . G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case . Thus elastic and dissipative stresses were coupled during changes in contractile activation . G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased . These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.

Biochim Biophys Acta, 2000 Jul 31, 1467(1), 85 - 90
Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells; Millot C et al.; We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro . After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis . Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.

Emerg Infect Dis, 2000 Sep-Oct, 6(5), 513 - 25
Using DNA microarrays to study host-microbe interactions; Cummings CA et al.; Complete genomic sequences of microbial pathogens and hosts offer sophisticated new strategies for studying host-pathogen interactions . DNA microarrays exploit primary sequence data to measure transcript levels and detect sequence polymorphisms, for every gene, simultaneously . The design and construction of a DNA microarray for any given microbial genome are straightforward . By monitoring microbial gene expression, one can predict the functions of uncharacterized genes, probe the physiologic adaptations made under various environmental conditions, identify virulence-associated genes, and test the effects of drugs . Similarly, by using host gene microarrays, one can explore host response at the level of gene expression and provide a molecular description of the events that follow infection . Host profiling might also identify gene expression signatures unique for each pathogen, thus providing a novel tool for diagnosis, prognosis, and clinical management of infectious disease.

Int J Parasitol, 2000 Sep, 30(10), 1115 - 21
The neurons of class ALD mediate thermotaxis in the parasitic nematode, Strongyloides stercoralis; Lopez PM et al.; Strongyloides stercoralis, a skin-penetrating nematode parasite of homeotherms, migrates to warmth . In nematodes, the amphids, anteriorly positioned, paired sensilla, each contain a bundle of sensory neurons . In the amphids of the free-living nematode Caenorhabditis elegans, a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors, and have been named the finger cells (class AFD) . A similar neuron pair in the amphids of the parasite Haemonchus contortus is also known to be thermosensory . Strongyloides stercoralis lacks finger cells but, in its amphids, it has a pair of neurons whose dendrites end in a multi-layered complex of lamellae, the so-called lamellar cells (class ALD) . Consequently, it was hypothesised that these lamellar cells might mediate thermotaxis by the skin-penetrating infective larva of this species . To investigate this, first stage S . stercoralis larvae were anaesthetised and the paired ALD class neurons were ablated with a laser microbeam . The larvae were then cultured to the infective third stage (L3) and assayed for thermotaxis on a thermal gradient . L3 with ablated ALD class neuron pairs showed significantly reduced thermotaxis compared with control groups . The thermoreceptive function of the ALD class neurons (i) associates this neuron pair with the host-finding process of S . stercoralis and (ii) demonstrates a functional similarity with the neurons of class AFD in C . elegans . The structural and positional characteristics of the ALD neurons suggest that these neurons may, in fact, be homologous with one pair of flattened dendritic processes known as wing cells (AWC) in C . elegans, while their florid development and thermosensory function suggest homology with the finger cells (AFD) of that nematode.

Anal Chem, 2000 Sep 1, 72(17), 4135 - 41
Rotating rod renewable microcolumns for automated, solid-phase DNA hybridization studies; Bruckner-Lea CJ et al.; The development of a new temperature-controlled renewable microcolumn flow cell for solid-phase nucleic acid hybridization in an automated sequential injection system is described . The flow cell included a stepper motor-driven rotating rod with the working end cut to a 45 degrees angle . In one position, the end of the rod prevented passage of microbeads while allowing fluid flow; rotation of the rod by 180 degrees releases the beads . This system was used to rapidly test many hybridization and elution protocols to examine the temperature and solution conditions required for sequence-specific nucleic acid hybridization . Target nucleic acids labeled with a near-infrared fluorescent dye were detected immediately postcolumn during all column perfusion and elution steps using a flow-through fluorescence detector . Temperature control of the column and the presence of Triton X-100 surfactant were critical for specific hybridization . Perfusion of the column with complementary oligonucleotide (200 microL, 10 nM) resulted in hybridization with 8% of the DNA binding sites on the microbeads with a solution residence time of less than 1 s and a total sample perfusion time of 40 s . The use of the renewable column system for detection of an unlabeled PCR product in a sandwich assay was also demonstrated.

Phonetica, 2000 Apr-Dec, 57(2-4), 128 - 38
The C/D model and prosodic control of articulatory behavior; Fujimura O; The Converter and Distributor (C/D) model is a generative description of articulatory gesture organization for utterances . Its input comprises specifications for syllables by features, a paraphonologically augmented metrical structure, and system parameters for utterance conditions . A syllable-boundary pulse train is computed as a time function representing the skeletal rhythmic structure of the utterance . Control functions for articulators are computed by superimposing consonantal elemental gestures onto the base function, which includes voicing, vocalic, mandibular, and tonal functions associated with the pulse train . A preliminary analysis of microbeam data for experimental dialogues, using jaw opening as an approximate measure of the syllable magnitude, inferred syllable and boundary durations consistently with durational characteristics observed in the acoustic waveform .

J Biomol Screen, 2000 Aug, 5(4), 269 - 78
Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates; Harms A et al.; A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter . MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters . Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h . Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact . Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50% . A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well . In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained . When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material . Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM {(3)H}ketanserin-HCl; at higher concentrations it was reached after about 10 min . Saturation analysis of {(3)H}ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source . Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2638 - 44
Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores; Kurosawa Y et al.; Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target . We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment) . The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter . The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds . Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines . Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity . Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity . Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model . These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

Nature, 2000 Aug 31, 406(6799), 989 - 92
The role of microbes in accretion, lamination and early lithification of modern marine stromatolites; Reid RP et al.; For three billion years, before the Cambrian diversification of life, laminated carbonate build-ups called stromatolites were widespread in shallow marine seas . These ancient structures are generally thought to be microbial in origin and potentially preserve evidence of the Earth's earliest biosphere . Despite their evolutionary significance, little is known about stromatolite formation, especially the relative roles of microbial and environmental factors in stromatolite accretion . Here we show that growth of modern marine stromatolites represents a dynamic balance between sedimentation and intermittent lithification of cyanobacterial mats . Periods of rapid sediment accretion, during which stromatolite surfaces are dominated by pioneer communities of gliding filamentous cyanobacteria, alternate with hiatal intervals . These discontinuities in sedimentation are characterized by development of surface films of exopolymer and subsequent heterotrophic bacterial decomposition, forming thin crusts of microcrystalline carbonate . During prolonged hiatal periods, climax communities develop, which include endolithic coccoid cyanobacteria . These coccoids modify the sediment, forming thicker lithified laminae . Preservation of lithified layers at depth creates millimetre-scale lamination . This simple model of modern marine stromatolite growth may be applicable to ancient stromatolites.

Appl Biochem Biotechnol, 2000 Jun, 87(3), 247 - 64
Development of an optimized feeding technology for dairy cows: improvement in resistance to ruminal proteases in the de novo-designed protein MB-1; Morrison JJ et al.; We have previously reported on MB-1, a designer protein with potential application in animal nutrition . Having a high content of selected essential amino acids, MB-1 should provide limiting nutrients for animals and promote growth and production . However, the protein was found to have marginal conformational and proteolytic stability, and, thus, strategies for stabilizing MB-1 were elaborated . We discuss the synthesis of MB-1-Cys dimer, a protein with an intermolecular disulfide bridge . This mutant was exposed to Pronase E protease preparation as well as to proteases extracted from ruminal microbes . It was found that in both cases, MB-1-Cys dimer had a better resistance to proteolytic degradation than MB-1 . Denaturation and hydrophobic dye binding studies revealed that this enhanced stability was not owing to conformational stabilization, but rather to changes in surface exposure as a consequence of dimerization . In particular, it was found that binding of ANSA to MB-1-Cys dimer was comparable to that observed for native, compact, natural proteins . We discuss the implications of these results for the design of transgenic protein production systems.

Int J Hematol, 2000 Jul, 72(1), 48 - 54
Rapid and prominent up-regulation of high-affinity receptor for immunoglobulin G (Fc gamma RI) by cross-linking of beta 2 integrins on polymorphonuclear leukocytes; Takano K et al.; Receptors for the Fc region (FcR) of immunoglobulin (Ig)G play essential roles in effector functions of polymorphonuclear leukocytes (PMNs) including the antibody-mediated clearance of microbes . In contrast to the constitutive expression of the low-affinity receptors for IgG (Fc gamma RII {CD32} and Fc gamma RIII {CD16}), the high-affinity receptor Fc gamma RI (CD64) is barely detectable on unactivated PMNs . CD64 expression is induced in a slow kinetic manner by interferon (IFN)-gamma and granulocyte colony-stimulating factor (G-CSF) after 12 to 24 hours of exposure to these agents . We found that the cross-linking of CD11b as well as of CD18 induced comparable rapid increases in CD64 expression on the surface of PMNs, occurring within 15 minutes of exposure . Cross-linking of neither CD11a nor CD11c induced CD64 expression . In contrast to slow induction by IFN-gamma and G-CSF, the integrin-induced rapid CD64 expression did not require RNA synthesis . Genistein, herbimycin A, and 1,2-bis(o-aminophenoxy)ethan-N,N-N',N'-tetraacetic acid blocked the immediate expression of CD64 in a dose-dependent manner, suggesting that the signal is mediated through calcium mobilization and protein tyrosine kinase(s) . Such rapid modulation of the high-affinity Fc gamma RI receptor by integrin cross-linking may reflect the requirement for rapid up-regulation of PMN effector functions, after interaction with endothelial cells, platelets or bacteria.

Indoor Air, 2000 Sep, 10(3), 138 - 45
An outbreak of respiratory diseases among workers at a water-damaged building--a case report; Seuri M et al.; We describe a military hospital building with severe, repeated and enduring water and mold damage, and the symptoms and diseases found among 14 persons who were employed at the building . The exposure of the employees was evaluated by measuring the serum immunoglobulin G (IgG)-antibodies against eight spieces of mold and yeast common in Finnish water and mold damaged buildings and by sampling airborne viable microbes within the hospital . The most abundant spieces was Sporobolomyces salmonicolor . All but one of the employees reported some building-related symptoms, the most common being a cough which was reported by nine subjects . Four new cases of asthma, confirmed by S . salmonicolor inhalation provocation tests, one of whom was also found to have alveolitis, were found among the hospital personnel . In addition, seven other workers with newly diagnosed rhinitis reacted positively in nasal S . salmonicolor provocation tests . Skin prick tests by Sporobolomyces were negative among all 14 workers . Exposure of the workers to mold and yeast in the indoor air caused an outbreak of occupational diseases, including asthma, rhinitis and alveolitis . The diseases were not immunoglobulin E (IgE)-mediated but might have been borne by some other, as yet unexplained, mechanism.

Cell Mol Biol (Noisy-le-grand), 2000 Sep, 46(6), 1053 - 63
Research at the European Synchrotron Radiation Facility medical beamline; Thomlinson W et al.; The application of synchrotron radiation in medical research has become a mature field of research at synchrotron facilities worldwide . In the relatively short time that synchrotrons have been available to the scientific community, their characteristic beams of UV and X-ray radiation have been applied to virtually all areas of medical science which use ionizing radiation . The ability to tune intense monochromatic beams over wide energy ranges differentiates these sources from standard clinical and research tools . At the European Synchrotron Radiation Facility (Grenoble, France), a major research facility is operational on an advanced wiggler radiation beamport, ID17 . The beamport is designed to carry out a broad range of research ranging from cell radiation biology to in vivo human studies . Medical imaging programs at ID17 include transvenous coronary angiography, computed tomography, mammography and bronchography . In addition, a major research program on microbeam radiation therapy is progressing . This paper will present a very brief overview of the beamline and the imaging and therapy programs.

Mol Plant Microbe Interact, 2000 Sep, 13(9), 942 - 50
Transgenic expression of pear PGIP in tomato limits fungal colonization; Powell AL et al.; Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development . Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP) . The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues . Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea . The growth of B . cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B . cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A . ten Have, W . Mulder, J . Visser, and J . A . L . van Kan, Mol . Plant-Microbe Interact . 11:1009-1016, 1998) . Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

Microbiol Mol Biol Rev, 2000 Sep, 64(3), 624 - 53
Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae-a pathogen, ice nucleus, and epiphyte; Hirano SS et al.; The extremely large number of leaves produced by terrestrial and aquatic plants provide habitats for colonization by a diversity of microorganisms . This review focuses on the bacterial component of leaf microbial communities, with emphasis on Pseudomonas syringae-a species that participates in leaf ecosystems as a pathogen, ice nucleus, and epiphyte . Among the diversity of bacteria that colonize leaves, none has received wider attention than P . syringae, as it gained notoriety for being the first recombinant organism (Ice(-) P . syringae) to be deliberately introduced into the environment . We focus on P . syringae to illustrate the attractiveness and somewhat unique opportunities provided by leaf ecosystems for addressing fundamental questions of microbial population dynamics and mechanisms of plant-bacterium interactions . Leaf ecosystems are dynamic and ephemeral . The physical environment surrounding phyllosphere microbes changes continuously with daily cycles in temperature, radiation, relative humidity, wind velocity, and leaf wetness . Slightly longer-term changes occur as weather systems pass . Seasonal climatic changes impose still a longer cycle . The physical and physiological characteristics of leaves change as they expand, mature, and senesce and as host phenology changes . Many of these factors influence the development of populations of P . syringae upon populations of leaves . P . syringae was first studied for its ability to cause disease on plants . However, disease causation is but one aspect of its life strategy . The bacterium can be found in association with healthy leaves, growing and surviving for many generations on the surfaces of leaves as an epiphyte . A number of genes and traits have been identified that contribute to the fitness of P . syringae in the phyllosphere . While still in their infancy, such research efforts demonstrate that the P . syringae-leaf ecosystem is a particularly attractive system with which to bridge the gap between what is known about the molecular biology of genes linked to pathogenicity and the ecology and epidemiology of associated diseases as they occur in natural settings, the field.

Dtsch Med Wochenschr, 2000 Aug 4, 125(31-32), 924 - 31
{Farmers' children suffer less from hay fever and asthma}; Gassner-Bachmann M et al.; BACKGROUND: Farm children suffer less frequently from pollinosis and bronchial asthma, although they are exposed to higher concentrations of airborne allergens . How do farm children differ from children in the general population with respect to immunological sensitization to airborne allergens? METHODS: Since 1983, a continuing seroepidemiological study of allergic reactions of schoolchildren in a rural area was conducted . Each year, all 15 year-old schoolchildren were given a standardized allergy questionnaire and are tested serologically (RAST/CAP-test) for their IgE sensitization towards the four major groups of airborne allergens: timothy grass pollen, birch pollen, house-dust mites, and cat dander . Farm children were compared with children who only occasionally help out on farms and also with those who had no direct contact with agriculture . RESULTS: From a total of 1307 children, 1287 questionnaires (98.5%) and 1100 (84.2%) of the serological tests could be analysed . Over the 16 years of the study, a statistically significant increase in the incidence and severity of hay fever and asthma was found in the children with no direct contact to agriculture . This was coupled with a significant increase in the sero-prevalence of sensitisation to the major airborne allergens . Among the 133 farm children, the prevalence of hay fever was: any history of hay fever in 2.4%, hay fever within the last year in none . A history of asthma was found in 1.6% of the children . Among children who occasionally worked on or had contact with farms, the prevalences were: any history of hay fever 9.3%, hay fever within the last year 9.3%, asthma 4.4% . Corresponding prevalences in children with no direct contact to farms were 18.3%, 18.6% and 9.1%, respectively . The seroepidemiological tests showed that the farm children not only had an overall lower rate of seroprevalence of antibodies towards the 4 marker allergens, but they also became sensitized to a lesser extent . At increasing cut-off values of the specific IgE titers, the odds ratio of the farm children in comparison to the control children became increasingly significant . This trend was found with all four marker allergens . The paradoxical immune reaction of farm children towards airborne allergens shows two characteristic quantitative features: in spite of high exposure rates, the children became sensitized less often and more weakly than control classmates from the same village . Children who had intermittent contact with farms showed intermediate results with respect to both symptoms and sensitization . CONCLUSIONS: These farm children have thus become adapted towards the allergens to which they are exposed . This immunological form ist similar to results from East Germany and the former USSR, in spite of the fact that lifestyle and exposure to microbes differ greatly . The immunological adaptation can best be interpreted as the result of a greater and continual stimulation of the Th1-immune response towards anthropozoonotic antigens.

Nature, 2000 Aug 17, 406(6797), 760 - 1
On the particularity of pathogens; Bloom BR; In the elemental struggle between pathogenic microbes and the immune system of the host, each strives for a unique advantage and thus each exploits its own unique particularities in pathogenesis and protection . And each presumably selects for the diversity that generally characterizes the wide range of successful host-pathogen interactions.

Xenotransplantation, 2000 May, 7(2), 83 - 92
Natural antibodies and the host immune responses to xenografts; Cramer DV; Natural antibodies are present in the serum of individuals in the absence of known antigenic stimulation . These antibodies are primarily IgM, polyreactive, and encoded by immunoglobulin V genes in germline configuration . Natural antibodies are produced by B-1 lymphocytes, cells that form the primary cell of the fetal and newborn B cell repertoire and may represent the basic foundation upon which the adult repertoire of B cell antibodies is based . Natural antibodies react with a variety of endogenous and exogenous antigens, including xenoantigens expressed by tissues between unrelated species . These antibodies are capable of causing the immediate rejection of grafts exchanged across species barriers . One of the central issues related to our understanding of the immunopathologic mechanisms responsible for rejection of xenografts is whether pre-formed natural antibodies and new antibodies induced following xenotransplantation are produced by the same pathways of B cell antibody production . We have established in studies conducted in rodents and humans that the initial phases of antibody production xenogeneic tissues involves the use of a restricted population of Ig germline genes to encode xenoantibody binding . As the humoral xenoantibody response matures, the same closely-related groups of Ig V genes are used to encode antibody binding and there is evidence for an isotype switch to IgG antibody production and the appearance of somatic mutations consistent with antigen-driven affinity maturation . Our findings in both rodent and human studies form the basis for our proposal that the xenograft response reflects the use of B cell natural antibody repertoires originally intended to provide protection against infection . The host humoral response is inadvertently recruited to mount antibody responses against foreign grafts because they display carbohydrate antigens that are shared by common environmental microbes . This model of xenoantibody responses is being tested in our laboratory through the analysis of the binding of xenoantibodies in their original non-mutated configuration, and the examination of the effect of specific point mutations and gene shuffling have on xenoantibody binding activity . Establishment of the relationships between Ig structural changes and subsequent changes in binding affinity should provide important insights into the role that, natural antibodies and the cells that produce them play in the evolution of the host's humoral responses to xenografts.

Org Lett, 2000 Aug 10, 2(16), 2401 - 4
Novel natural products from soil DNA libraries in a streptomycete host; Wang GY et al.; As a route to accessing the potential chemical diversity of uncultivable microbes from the soil, combinatorial biosynthetic libraries were constructed by cloning large fragments of DNA isolated from soil into a Streptomyces lividans host . Four novel compounds, terragines A (1), B (2), C (3), and D (4), were isolated from recombinant 436-s4-5b1, and another novel compound, terragine E (5), was isolated from 446-s3-102g1 . The structures were determined by a combination of spectroscopic techniques, primarily 2D NMR.

Int J Cancer, 2000 Jul 20, 89(4), 337 - 44
Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer; Iinuma H et al.; A new method for detecting circulating tumor cells that is based on magnetic-activated cell separation (MACS) and nested mutant allele-specific amplification (nested MASA) was evaluated in patients with colorectal cancer using the p53 and K-ras genes as genetic markers . By negative selection with anti-CD45 monoclonal antibody-conjugated supermagnetic microbeads, the proportion of tumor cells was enriched 9-fold . By the combination of MACS and nested MASA, 10 tumor cells in 10(7) normal peripheral blood mononuclear cells could be detected without false-positives . Using this method, we examined blood taken from the tumor drainage veins of 23 patients with colorectal cancer . Eighty-seven percent (20/23) of primary tumor tissues showed p53 and/or K-ras gene mutations . Forty-five percent (9/20) of patients with p53 and/or K-ras mutations in the primary tumor showed the same mutated genes in the blood samples . There was a significant association between the presence of p53 and K-ras gene mutation in the blood and tumor size, depth of invasion, and venous invasion . Blood gene mutation was detected in 80% (4/5) of samples from patients with synchronous liver metastases . Sixty percent (3/5) of patients with mutant genes in the blood developed asynchronous liver metastases after surgery . The overall survival of patients with p53 and/or K-ras gene mutation-positive findings in blood was significantly shorter than that of patients testing negative on Kaplan-Meier analysis . Our results suggest that the method may be useful for reliable detection of tumor cells circulating in the blood and may help to identify patients at high risk for relapse .

Med Phys, 2000 Jul, 27(7), 1664 - 75
Physics study of microbeam radiation therapy with PSI-version of Monte Carlo code GEANT as a new computational tool; Stepanek J et al.; Microbeam radiation therapy (MRT) is a currently experimental method of radiotherapy which is mediated by an array of parallel microbeams of synchrotron-wiggler-generated x-rays . Suitably selected, nominally supralethal doses of x-rays delivered to parallel microslices of tumor-bearing tissues in rats can be either palliative or curative while causing little or no serious damage to contiguous normal tissues . Although the pathogenesis of MRT-mediated tumor regression is not understood, as in all radiotherapy such understanding will be based ultimately on our understanding of the relationships among the following three factors: (1) microdosimetry, (2) damage to normal tissues, and (3) therapeutic efficacy . Although physical microdosimetry is feasible, published information on MRT microdosimetry to date is computational . This report describes Monte Carlo-based computational MRT microdosimetry using photon and/or electron scattering and photoionization cross-section data in the 1 eV through 100 GeV range distributed publicly by the U.S . Lawrence Livermore National Laboratory (LLNL) in the 1990s . These are compared with Monte Carlo-based microdosimetric computations using a code and physical data available in the 1980s . With the aim of using the PSI-version of GEANT Monte Carlo code for future macro- and micro/nano-dosimetric studies of Microbeam Radiation Therapy (MRT) a comparison of this code is made with the INHOM(EGS4) (version 1990), Dilmanian-CPE and Persliden-CPE Monte Carlo photon-electron codes (both version 1990) with which the absorbed dose distributions were calculated in 1990 and 1991 considering, (a) a single cylindrical microbeam, (b) multiple cylindrical microbeams in an orthogonal square bundle, and (c) multiple planar microbeams . It is shown that the PSI-version of GEANT can potentially deliver more accurate results (a) using presently the most advanced atomic data, and especially (b) employing "Single-collision" electron transport instead of only the "Condensed-history" electron transport as in code INHOM(EGS4) . In contrast Dilmanian-CPE and Persliden-CPE codes deposit the electron energy locally instead of transporting it to the correct position.

Int J Food Microbiol, 2000 Jul 25, 59(1-2), 1 - 7
Degradation of ochratoxin A by Aspergillus species; Varga J et al.; Mycotoxin contamination of agricultural products is a serious health hazard throughout the world . Besides attempts to eliminate mycotoxins from contaminated substrates by physical and chemical methods, the ability of microbes to degrade mycotoxins is now being widely examined . In this study, several Aspergillus species were examined for their ability to degrade ochratoxin A . A . fumigatus and black Aspergillus strains were found to detoxify ochratoxin A in culture media . The kinetics of ochratoxin A detoxification by an atoxigenic A . niger strain was examined by thin layer chromatography, high-performance liquid chromatography and an immunochemical technique . A . niger CBS 120.49 was found to effectively eliminate ochratoxin A from both liquid and solid media, and the degradation product, ochratoxin alpha, was also decomposed.

Biosens Bioelectron, 2000 Jan, 14(10-11), 805 - 13
The BARC biosensor applied to the detection of biological warfare agents; Edelstein RL et al.; The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents . The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet . DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface . Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA . A magnetic field is applied, removing any beads that are not specifically bound to the surface . The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample . The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes . Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.

Plant Cell Physiol, 2000 Jun, 41(6), 726 - 32
Isolation of two different phenotypes of mycorrhizal mutants in the model legume plant Lotus japonicus after EMS-treatment; Senoo K et al.; Lotus japonicus has been proposed as a model plant for the molecular genetic study of plant-microbe interaction including Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi . Non-mycorrhizal mutants of Lotus japonicus were screened from a collection of 12 mutants showing non-nodulating (Nod-), ineffectively nodulating (Fix-) and hypernodulating (Nod++) phenotypes with monogenic recessive inheritance induced by EMS (ethylmethane sulfonate) mutagenesis . Three mycorrhizal mutant lines showing highly reduced arbuscular mycorrhizal colonization were obtained . All of them were derived from Nod- phenotypes . In Ljsym72, the root colonization by Glomus sp . R-10 is characterized by poor development of the external mycelium, formation of extremely branched appressoria, and the blocking of hyphal penetration at the root epidermis . Neither arbuscules nor vesicles were formed in Ljsym72 roots . Fungal recognition on the root surface was strongly affected by the mutation in the LjSym72 gene . Unique characteristics in mutant lines Ljsym71-1 and Ljsym71-2 were the overproduction of deformed appressoria and arrested hyphal penetration of the exodermis . Small amounts of internal colonization including degenerated arbuscule formation occurred infrequently in these types of mutants . Not only fungal development on the root surface but also that in the root exodermis and cortex was affected by the mutation in LjSym71 gene . These mutants represent a key advance in molecular research on the AM symbiosis.

Springer Semin Immunopathol, 2000, 22(1-2), 97 - 105
Signal transduction induced by immunostimulatory CpG DNA; Krieg AM; The immune recognition of unmethylated CpG motifs appears to be an example of the ability of the immune system to detect molecular patterns which are characteristic of microbes, but are not present in vertebrates . This detection is accomplished by the means of pattern recognition receptors (PRR) . Unlike some other examples of PRR, immune recognition of CpG DNA appears to require cell uptake and to be accomplished through an intracellular PRR system . This then results in the activation of mitogen-activated protein kinases, culminating in the phosphorylation of transcription factors and the activation of transcription and translation . The rapid activation of these pathways by CpG DNA leads to the induction of protective immune responses.

Women Health, 2000, 30(3), 93 - 110
The social and medical construction of lactation pathology; Wolf JH; Beginning in the 1880s, many mothers reported breastfeeding difficulties . Doctors blamed the stress of urban life . The "bad" human milk invariably produced by the mammary glands of urban women, some physicians charged, harmed babies as surely as the dirty and adulterated cow's milk common to the late nineteenth-century city . Mothers and pediatricians proved unusually susceptible to believing this allegation . Mothers, just learning about the germ theory of disease and anxious about protecting their babies from unseen microbes, found themselves gratefully relying on "scientific" food rather than on their own, apparently faulty, bodies . And pediatricians no longer had to defend their new specialty . Now they could point to the need for improved artificial food-given women's growing inability to lactate-as one justification for their specialty's existence . Under the influence of these mothers and doctors, the notion that human lactation is an unreliable body function became a cultural truth that has persisted unabated to the present day.

Annu Rev Nutr, 2000, 20, 699 - 722
Oligosaccharides in human milk: structural, functional, and metabolic aspects; Kunz C et al.; Research on human milk oligosaccharides (HMOs) has received much attention in recent years . However, it started about a century ago with the observation that oligosaccharides might be growth factors for a so-called bifidus flora in breast-fed infants and extends to the recent finding of cell adhesion molecules in human milk . The latter are involved in inflammatory events recognizing carbohydrate sequences that also can be found in human milk . The similarities between epithelial cell surface carbohydrates and oligosaccharides in human milk strengthen the idea that specific interactions of those oligosaccharides with pathogenic microorganisms do occur preventing the attachment of microbes to epithelial cells . HMOs may act as soluble receptors for different pathogens, thus increasing the resistance of breast-fed infants . However, we need to know more about the metabolism of oligosaccharides in the gastrointestinal tract . How far are oligosaccharides degraded by intestinal enzymes and does oligosaccharide processing (e.g . degradation, synthesis, and elongation of core structures) occur in intestinal epithelial cells? Further research on HMOs is certainly needed to increase our knowledge of infant nutrition as it is affected by complex oligosaccharides.

Anal Chem, 2000 Jul 15, 72(14), 3355 - 62
Protein concentration and enzyme digestion on microbeads for MALDI-TOF peptides mass mapping of proteins from dilute solutions; Doucette A et al.; A method for generating peptide mass maps from dilute protein samples is presented . The method involves the concentration of proteins from aqueous solution by adsorption onto reversed-phase polymeric microbeads . These beads are then washed extensively to remove contaminants, after which the bound proteins are digested with trypsin . Analysis of the digestion products is performed by MALDI-TOF mass spectrometry following direct deposition of the beads on a MALDI target, along with the matrix solution . The procedure is demonstrated using solutions of cytochrome c, lysozyme, and bovine serum albumin . The results of these digests are compared to trypsin digestions of the protein samples without sample preconcentration . Comparative results are also presented for protein solutions contaminated with 2 M NaCl, 2 M urea, or sodium dodecyl sulfate at concentrations up to 0.02% . These results reveal that, with the microbead preconcentration procedure, peptide mass maps can routinely be generated from highly contaminated samples with a protein concentration of only 100 nM.

Anal Chem, 2000 Jul 15, 72(14), 3109 - 15
Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy; Hodder PS et al.; Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay . Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands . A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber . The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system . The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine . In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response . Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

Mol Plant Microbe Interact, 2000 Aug, 13(8), 830 - 6
An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis; Qin L et al.; A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis . Three independent criteria were used for selection . First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages . For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism) . Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles . This was checked by in situ hybridization . As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion . Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified . No dorsal gland genes have been cloned before from plant-parasitic nematodes . The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene . Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses . Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Toxicon, 2001 Jan, 39(1), 87 - 96
Toxicity in animals . Trends in evolution?
Mebs D.
Animals acquire toxicity either by metabolic synthesis of toxins (secondary metabolites), by expression of toxin genes or by the uptake, storage and sequestration of toxins produced by other organisms, i.e., microbes, plants or other animals . Variability of toxin structure and function is high . Peptide toxins in particular, although relying on a limited number of structural frameworks, often exhibit considerable structural hypervariability . An accelerated rate of evolution in the toxin gene structure (conserved introns, but high substitution rates in the exons) leads to the functional diversity of these peptides or proteins . The selective forces which may drive toxin evolution are unknown . Venomousness or the possession of toxins can be essential for survival, but the advantage of toxin biosynthesis may also be of minor importance or has been lost during evolution.

Mol Ther, 2000 Jan, 1(1), 82 - 7
Angiogenesis monitored by perfusion with a space-filling microbead suspension; Springer ML et al.; Numerous laboratories are focusing efforts on delivering gene products to induce or prevent the development of new blood vessels in adults, with the hope of rescuing ischemic tissues, circumventing cardiac bypass surgery, or inhibiting tumor growth . Current approaches to the assessment of vascular continuity involve the introduction of either dyes or fluorescent microspheres to track blood flow . However, dyes and dextrans are subject to leakage when vessels are hyperpermeable, a situation that may occur in studies of tumor vasculature and during efforts to stimulate therapeutic angiogenesis . Furthermore, the microspheres that are used for flow studies do not allow a comprehensive visual analysis of vascular continuity . Here we report a method for the visual assessment of microvascular continuity in mouse muscle under circumstances in which vessels are leaky . The approach involves perfusion of the vasculature with fluorescent beads that are much smaller than those used for flow studies . The suspension behaves like a fluid and completely fills the vessels, yet the beads do not leak from VEGF-permeablized capillaries and remain localized in histological sections . Use of beads with the proper fluorescence emission wavelengths allows immunofluorescent colocalization with vessel-specific markers . We compare this improved method with other methods for tracking vascular continuity involving dextrans and larger beads . This approach should aid in the dynamic study of tumor angiogenesis and the evaluation of efforts to deliver angiogenic factors.

Immunol Rev, 2000 Jun, 175, 214 - 28
Development of the antibody repertoire in rabbit: gut-associated lymphoid tissue, microbes, and selection; Lanning D et al.; Rabbits generate their antibody repertoire in three stages . First, a neonatal repertoire is generated by B lymphopoiesis in fetal liver and bone marrow and is limited by preferential V(H) gene segment usage . Between 4 and 8 weeks after birth a complex primary antibody repertoire is developed by somatically diversifying the neonatal repertoire through somatic hypermutation and a somatic gene conversion-like mechanism in gut-associated lymphoid tissue (GALT) . In rabbits, unlike other species, the development of the primary antibody repertoire through somatic diversification of Ig genes appears to be dependent on intestinal microbial flora . The primary antibody repertoire is subsequently modified during antigen-dependent immune responses in which VDJ genes further diversify both by somatic hypermutation and by a gene conversion-like mechanism (the secondary repertoire) . During the various stages of development, the antibody repertoire is modified and shaped by selective processes . In this review, we discuss the roles of GALT, microbes, and B-cell selection in generating antibody diversity in rabbits.

Microbiology, 2000 Aug, 146 ( Pt 8), 2079 - 89
Induction of the sexual stage of Pestalotiopsis microspora, a taxol-producing fungus; Metz AM et al.; Pestalotiopsis microspora, isolate NE-32, is an endophyte of the Himalayan yew (Taxus wallichiana) that produces taxol, an important chemotherapeutic drug used in the treatment of breast and ovarian cancers . Conditions were determined to induce the perfect stage (teleomorph) of this organism in the laboratory as a critical first step to study inheritance of taxol biosynthetic genes . The perfect stage of Pestalotiopsis microspora NE-32 forms in a period of 3-6 weeks on water agarose with dried yew needles at 16-20 degrees C with 12 h of light per day . Morphological analysis of the teleomorph and sequencing of the 18S rDNA indicates that Pestalosphaeria hansenii is the perfect stage of Pestalotiopsis microspora . Only certain plants (e.g . yews, some pines, pecan, oat and some barley cultivars) allow the production of perithecia . Exhaustive methylene chloride extraction of yew (Taxus cuspidata) needles removes their capacity to induce production of perithecia . The methylene chloride extract is able to induce formation of perithecia by strain NE-32 in a bioassay system utilizing the sterilized sheaths of the Cholla cactus (Opuntia bigelovii) spine, indicating that a chemical compound(s) in yew stimulates the formation of the perfect stage . This hydrophobic plant compound(s) has been designated the perithecial-stimulating factor (PSF) . The data suggest that plant products may play a role in regulating the biology of endophytic microbes.

Immunology, 2000 Aug, 100(4), 411 - 6
Review article: ageing and the neutrophil: no appetite for killing?
Butcher S, Chahel H, Lord JM.
In the armoury of the immune system developed to combat the various micro-organisms that could invade the host, the neutrophil forms the first line of defence against rapidly dividing bacteria and fungi . However, as humans age they become more susceptible to infection with these microbes and this has been ascribed to a decline in immune status, termed immune senescence . Here we summarize the literature specifically concerning the attenuation of neutrophil function with age and the possible mechanisms underlying their reduced response to infectious agents.

Dev Biol, 2000 Aug 15, 224(2), 339 - 53
Leech segmental repeats develop normally in the absence of signals from either anterior or posterior segments; Seaver EC et al.; We have investigated whether the development of segmental repeats is autonomous in the embryo of the leech Helobdella robusta . The segmental tissues of the germinal band arise from progeny of five stem cells called teloblasts . Asymmetric divisions of the teloblasts form chains of segment founder cells (called primary blast cells) that divide in a stereotypical manner to produce differentiated descendants . Using two distinct techniques, we have looked for potential interactions between neighboring blast cell clones along the anterior-posterior axis . In one technique, we prevented the birth of primary blast cells by injection of DNase I into the teloblast, thereby depriving the last blast cell produced before the ablation of its normal posterior neighbors . We also ablated single blast cells with a laser microbeam, which allowed us to assess potential signals acting on either more anterior or more posterior primary blast cell clones . Our results suggest that interactions along the anterior-posterior axis between neighboring primary blast cell clones are not required for development of normal segmental organization within the blast cell clone . We also examined the possibility that blast cells receive redundant signals from both anterior and posterior neighboring clones and that either is sufficient for normal development . Using double blast cell laser ablations to isolate a primary blast cell clone by removal of both its anterior and its posterior neighbor, we found that the isolated clone still develops normally . These results reveal that the fundamental segmental repeat in the leech embryo, the primary blast cell clone, can develop normally in the apparent absence of signals from adjacent repeats along the anterior-posterior axis.

J Immunol, 2000 Aug 15, 165(4), 2124 - 33
Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages; Hu B et al.; Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice . Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMphi) clear apoptotic lymphocytes poorly . To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMphi and peritoneal macrophages (PMphi) from normal C57BL/6 mice . AMphi were deficient relative to PMphi both in percentage containing apoptotic thymocytes (19.1 +/- 1% vs 96 +/- 2.6% positive) and in phagocytic index (0.23 +/- 0.02 vs 4.2 +/- 0.67) . This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads . Annexin V blockade indicated that both Mphi types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression . By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage . To confirm these studies, apoptotic thymocytes were given intratracheally or i.p . to normal mice, and then AMphi or PMphi were recovered 30-240 min later . Ingestion of apoptotic thymocytes by AMphi in vivo was significantly decreased at all times . Defective ingestion of apoptotic lymphocytes may preserve AMphi capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.

Appl Environ Microbiol, 2000 Aug, 66(8), 3214 - 20
Microbial life beneath a high arctic glacier; Skidmore ML et al.; The debris-rich basal ice layers of a high Arctic glacier were shown to contain metabolically diverse microbes that could be cultured oligotrophically at low temperatures (0.3 to 4 degrees C) . These organisms included aerobic chemoheterotrophs and anaerobic nitrate reducers, sulfate reducers, and methanogens . Colonies purified from subglacial samples at 4 degrees C appeared to be predominantly psychrophilic . Aerobic chemoheterotrophs were metabolically active in unfrozen basal sediments when they were cultured at 0.3 degrees C in the dark (to simulate nearly in situ conditions), producing (14)CO(2) from radiolabeled sodium acetate with minimal organic amendment (> or =38 microM C) . In contrast, no activity was observed when samples were cultured at subfreezing temperatures (< or =-1.8 degrees C) for 66 days . Electron microscopy of thawed basal ice samples revealed various cell morphologies, including dividing cells . This suggests that the subglacial environment beneath a polythermal glacier provides a viable habitat for life and that microbes may be widespread where the basal ice is temperate and water is present at the base of the glacier and where organic carbon from glacially overridden soils is present . Our observations raise the possibility that in situ microbial production of CO(2) and CH(4) beneath ice masses (e.g., the Northern Hemisphere ice sheets) is an important factor in carbon cycling during glacial periods . Moreover, this terrestrial environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap.

Cytometry, 2000 Aug 1, 40(4), 307 - 15
Magnetophoretic mobilities correlate to antibody binding capacities; McCloskey KE et al.; METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology . RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles . Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient . DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient . A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained . CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC . The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells .

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 51 - 6
Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var . nicotianae; Bottin A et al.; PEG-mediated transformation of protoplasts in the presence of lipofectin was achieved in Phytophthora parasitica var . nicotianae, an oomycete pathogen of tobacco . Using oomycete promoter and terminator sequences, a plant-adapted green fluorescent protein (GFP) was introduced into the microorganism . The data show for the first time that this eukaryotic gene reporter can be used in an oomycete, both as a quantitative reporter of gene induction and as a vital marker allowing the study of development of Phytophthora in vitro and in the host plant.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8841 - 8
Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens; Nathan C et al.; This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity . Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense . In combination, the contribution of these two enzymes appears to be greater than previously appreciated . The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI . Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role . In diseases such as tuberculosis, a molecular understanding of host-pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates . Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8799 - 806
Exploitation of host cells by enteropathogenic Escherichia coli; Vallance BA et al.; Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells . One such subversive microbe is enteropathogenic Escherichia coli (EPEC) . A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide . Central to EPEC-mediated disease is its colonization of the intestinal epithelium . After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions . Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease . Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir) . Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells . The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria . This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions . Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

J Immunol Methods, 2000 Jul 31, 241(1-2), 97 - 108
Dependence of T cell activation on area of contact and density of a ligand-coated surface; Patrick SM et al.; An apparatus which allows precise control of the time of initiation and the area of contact of cells with immobilized ligands has been developed . Cells are trapped in an asymmetric film that can be quantitatively thinned, forcing the cells into close contact with ligands adsorbed on the base of the apparatus . Using microbeads to indicate the film height, the amount of thinning can be controlled to within 1 microm, producing known contact areas between cells and the ligand-coated surface . This system was used with anti-CD3-coated surfaces of different densities to examine the effect of ligand density on T cell activation, while keeping the number of ligands presented to the cells constant . T cell activation was observed individually in each cell as intracellular calcium mobilization . In these experiments both the percent of T cell activation and the rate of calcium rise were found to depend only on the number of anti-CD3 molecules presented and not on their density . This implies that the spacing between molecules is not important in the range studied, and suggests that receptor clustering to levels higher than dimers may not be necessary for induction of calcium mobilization by anti-CD3.

Crit Rev Biochem Mol Biol, 2000, 35(3), 221 - 51
Structural and functional comparison of polysaccharide-degrading enzymes; Jedrzejas MJ; Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates . Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space . Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes . Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues . Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a beta-elimination process . The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography . There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes . The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action . The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.

Adv Microb Physiol, 2000, 43, 1 - 38
Metal ion transport in eukaryotic microorganisms: insights from Saccharomyces cerevisiae; Eide DJ; Metal ions such as iron, copper, manganese, and zinc are essential nutrients for all eukaryotic microorganisms . Therefore, these organisms possess efficient uptake mechanisms to obtain these nutrients from their extracellular environment . Metal ions must also be transported into intracellular organelles where they function as catalytic and structural cofactors for compartmentalized enzymes . Thus, intracellular transport mechanisms are also present . When present in high levels, metal ions can also be toxic, so their uptake and intracellular transport is tightly regulated at both transcriptional and post-transcriptional levels to limit metal ion overaccumulation and facilitate storage and sequestration . Remarkable molecular insight into these processes has come from recent studies of the yeast Saccharomyces cerevisiae . This organism, which is the primary subject of this chapter, serves as a useful paradigm to understand metal ion metabolism in other eukaryotic microbes.

EXS, 1999, 87, 157 - 69
Aggressive and defensive roles for chitinases; Gooday GW; Chitinases are produced by a wide variety of pathogenic and parasitic microbes and invertebrates during their attack on chitin-containing organisms . Examples discussed include enzymes of insect and algal viruses, of yeast killer toxin plasmids, of bacterial and fungal pathogens of fungi and insects, and of parasitic protozoa . These chitinases play roles in penetration of fungal cell walls, and of exoskeletons and peritrophic membranes of arthropods . Salivas of some invertebrate predators have chitinolytic activity which may be involved in their attack on their prey . Chitinases play a major defensive role in all plants against attack by fungi, and perhaps also against attack by insect pests . The plant chitinases form a very large and diverse assemblage of enzymes from two families of glycosyl hydrolases . At least some vertebrates, including fish and humans, also may utilise chitinases in their defence against pathogenic fungi and some parasites.

J Immunol, 2000 Aug 1, 165(3), 1486 - 90
Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin; Wang Z et al.; Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes . Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions . In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity . Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic . Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence . Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets . While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells . In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions . Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides . Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

Comb Chem High Throughput Screen, 2000 Jun, 3(3), 185 - 96
Recombinant polyclonal antibody libraries; Sharon J et al.; We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen) . Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations . The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries . This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies . We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

Chemosphere, 1999 Jan, 38(1), 143 - 54
Behaviour of EDTA in marine microcosms
Virtapohja J, Alen R.
Laboratory-scale microcosm tests were carried out in sea water with and without sediment to investigate the importance of ultraviolet (UV) light and microbes in the temperature-dependent degradation of metal complexes of ethylenediaminetetraacetic acid (EDTA) . After 17 weeks, 44% and 48% of the original EDTA (initial concentration 385 microg/L) were converted at 10 degrees C and 22 degrees C, respectively . The degradation was more influenced by UV light than by sediment microbes, although the latter were very important . At both temperatures, absorption of EDTA to the sediment phase reached a maximum in the initial stage of the experiments (<4 weeks): at 10 degrees C about 4% and at 22 degrees C about 7% of the original EDTA.

Int J Food Microbiol, 2000 Jun 30, 58(1-2), 39 - 48
Hygiene aspects of modern poultry chilling; Allen VM et al.; An evaluation was made of six commercial poultry chilling systems in relation to factors affecting microbial-contamination of carcasses . These systems included water immersion chilling, air chilling and air chilling with evaporative cooling using water sprays . Samples of neck skin and body cavity were taken from carcasses, together with samples from the chilling environment . These were examined for total aerobic mesophilic microbes and counts of presumptive coliform bacteria and Pseudomonas spp . at specific points in the chilling process . Physical measurements included surface and deep-muscle temperatures of carcasses, water temperatures and chlorine concentrations in the immersion system and air speed and temperature during air chilling . The results obtained for water immersion chilling confirmed previous experience that the washing effect reduces microbial contamination of carcasses, although initially the numbers of pseudomonads tended to increase . The air chillers varied in design and mode of operation, but had little overall effect on microbial contamination of the skin . When a completely dry process was used, microbial numbers were reduced approximately ten-fold in the body cavity . However, the use of water sprays tended to increase contamination of the cavity, while relatively heavy spraying using non-chlorinated water, resulted in a substantial increase in the numbers of pseudomonads.

Nature, 2000 Jul 6, 406(6791), 54 - 6
Phosphate concentrations in lakes; Hudson JJ et al.; Phosphate is an important nutrient that restricts microbial production in many freshwater and marine environments . The actual concentration of phosphate in phosphorus-limited waters is largely unknown because commonly used chemical and radiochemical techniques overestimate the concentration . Here, using a new steady-state radiobioassay to survey a diverse set of lakes, we report phosphate concentrations in lakes that are orders of magnitude lower than estimates made spectrophotometrically or with the frequently used Rigler radiobioassay . Our results, combined with those from the literature, indicate that microbes can achieve rapid turnover rates at picomolar nutrient concentrations . This occurs even though these concentrations are about two orders of magnitude below the level where phosphate uptake is estimated to be half the saturation level for the pico-plankton community . Also, while phosphate concentration increased with the concentration of total phosphorus and soluble reactive phosphorus in the lakes we sampled, the proportion of phosphate in the total phosphorus pool decreased from oligotrophic to eutrophic lakes . Such information, as revealed by the phosphate assay that we use here, should allow us to address hypotheses concerning the concentration of phosphate available to planktonic microorganisms in aquatic systems.

Proc Natl Acad Sci U S A, 2000 Jul 18, 97(15), 8445 - 50
CD1c molecules broadly survey the endocytic system; Sugita M et al.; The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection . Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments . In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens . Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments . Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification . Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

J Comp Neurol, 2000 Aug 14, 424(1), 58 - 73
Thermotaxis and thermosensory neurons in infective larvae of Haemonchus contortus, a passively ingested nematode parasite; Li J et al.; As a basis for studies of thermal behavior of infective larvae (L3) of Haemonchus contortus resulting from ablation of amphidial neurons, the locations of the amphidial cell bodies in the hatchling larva (L1) were compared with their locations in the L3 . We sought to verify that killing each targeted cell body in L1 destroys the putative corresponding dendrite of the L3 . These comparisons confirmed the predicted cell body-to-dendrite connections, as well as similarities in the general amphidial structure of the two stages . We then conducted a series of studies using laser microbeam ablation of amphidial cell bodies in the L1 to determine the role of specific neurons in the thermal behavior of the L3 . In a thermal gradient, normal L3 of H . contortus migrate to the temperature at which they were cultured and/or maintained . Larvae grown at 16 degrees or 26 degrees C migrate appropriately to either of these temperatures . Larvae grown to the L3 stage at 16 degrees C and then moved to 26 degrees C become acclimated to this temperature and thereafter migrate to it . However, when the putative thermosensory neurons, the finger cell neurons (AFD), were ablated in hatchling larvae with a laser microbeam, and these were grown to the L3 stage and tested on a radial thermal gradient, they failed to migrate to their culture temperature . Instead, they moved actively and continuously over much of the assay plate surface, with no obviously oriented cryo- or thermotactic movement . Ablation-control larvae, those in which putatively chemosensory neuron classes ASE or AWC were killed, migrated normally to their culture temperature . When the RIA interneurons (identified by positional homology with those of Caenorhabditis elegans) were ablated, the operated larvae moved actively, but circled near the initial placement point; control larvae, in which other nonamphidial neurons were killed, migrated normally . These results indicate that the finger cell neurons (AFD) are the primary thermosensory class in H . contortus . The RIA-class neurons integrate thermal responses in H . contortus, as do their putative structural homologs in C . elegans, but the behavior of H . contortus subsequent to RIA ablation is strikingly different .

Yi Chuan Xue Bao, 2000, 27(3), 278 - 82
{Bacterial genetic diversity in soils and their correlation with vegetation}; Yang GP et al.; Soil is an important epicenter of biodiversity . Human activity has threatened such biodiversity through vegetation shift . As the major type of microbes, bacteria have played key roles in material cycling . Since simple morphological characters and the failure of the major portion of bacteria in surviving artificial cultivation have hindered bacterial diversity from analyses using traditional taxonomic approaches, bacterial diversity and its correlation with vegetation is less understood . In order to detour cultivation, tag serial sequencing strategy was developed in this study, in which a 26 base pair highly variable region has been chosen from 16S ribosomal RNA gene as variant-representing tags, ligated into serials and sequenced . Molecular bacterial diversity in soils has been determined using tag types, tag frequencies and diversity index as parameters . The correlation of bacterial diversity with vegetation has been explored as well . Bacterial diversity and differentiation correlate highly with contents of organic matter and nitrogen of soils and further vegetation.

Infection, 1999, 27(4-5), 252 - 5
Helicobacter pylori--a trigger of reactive arthritis?
Melby KK, Kvien TK, Glennas A.
Four cases of reactive arthritis (ReA) related to Helicobacter pylori (HP) are presented . These were identified by IgG, IgM and IgA ELISA tests performed on sera obtained from a 2-year prospective study on 186 patients with a clinical picture suggesting ReA as a possible diagnosis . If anti-HP IgM and IgA or IgG were positive, the case was considered related to HP.Three out of four HP ReA patients were originally classified as "possible ReA", i.e . having a clinical picture of ReA but without any identified triggering microorganism . IgG antibodies against cagA and vacA were detected in three and two cases respectively.The HP ReA patients did not present with typical clinical or laboratory features differentiating them from ReA induced by Chlamydia trachomatis (N = 25) or enteropathogenic bacteria (N = 27) . However, compared to findings in patients with ReA due to enteropathogenic bacteria the number of active joints was higher (six versus two), duration of arthritis longer (3.9 weeks versus 2 weeks) and the CRP (C-reactive protein) lower (43 versus 59) . Our findings suggest that HP may be included in the list of possible arthritis triggering microbes.

EMBO J, 2000 Jul 3, 19(13), 3325 - 36
The apoptotic signaling pathway activated by Toll-like receptor-2; Aliprantis AO et al.; The innate immune system uses Toll family receptors to signal for the presence of microbes and initiate host defense . Bacterial lipoproteins (BLPs), which are expressed by all bacteria, are potent activators of Toll-like receptor-2 (TLR2) . Here we show that the adaptor molecule, myeloid differentiation factor 88 (MyD88), mediates both apoptosis and nuclear factor-kappaB (NF-kappaB) activation by BLP-stimulated TLR2 . Inhibition of the NF-kappaB pathway downstream of MyD88 potentiates apoptosis, indicating that these two pathways bifurcate at the level of MyD88 . TLR2 signals for apoptosis through MyD88 via a pathway involving Fas-associated death domain protein (FADD) and caspase 8 . Moreover, MyD88 binds FADD and is sufficient to induce apoptosis . These data indicate that TLR2 is a novel 'death receptor' that engages the apoptotic machinery without a conventional cytoplasmic death domain . Through TLR2, BLP induces the synthesis of the precursor of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) . Interestingly, BLP also activates caspase 1 through TLR2, resulting in proteolysis and secretion of mature IL-1beta . These results indicate that caspase activation is an innate immune response to microbial pathogens, culminating in apoptosis and cytokine production.

Dig Dis Sci, 2000 Jun, 45(6), 1121 - 9
Is Crohn's disease an immunodeficiency? A hypothesis suggesting possible early events in the pathogenesis of Crohn's disease; Korzenik JR et al.; The current hypothesis for the etiology of Crohn's disease proposes an excessive immune response, largely T-cell driven, possibly against endogenous bacteria . Standard therapy is therefore directed towards suppression of this immune response . An alternative theory of pathogenesis accounts for epidemiologic and pathophysiologic observations that have been hitherto underemphasized, namely, (1) genetic disorders with deficiencies in neutrophil function can give rise to a clinical and pathologic syndrome indistinguishable from Crohn's; (2) abnormal neutrophil function is well described in Crohn's disease; (3) a group of bacteria implicated in other chronic inflammatory disorders causes impairment of neutrophil function; and (4) 20th century environmental risk factors for Crohn's disease may directly suppress neutrophil function and may have led to a shift in the dominant gut flora with similar effects . We propose that some cases of Crohn's disease result from the interaction of environmental and genetic influences leading to impaired mucosal neutrophil function, resulting in failure to effectively clear intramucosal microbes effectively . While encompassing existing data, this hypothesis proposes a proximate defect in the mucosal immune response . If this paradigm were correct, new therapeutic approaches might involve strategies to alter intestinal flora and stimulate neutrophil function.

J Photochem Photobiol B, 2000 Mar, 55(1), 16 - 9
Triplet-state photophysics of aluminium phthalocyanine sensitizer in murine cancer cells; Oldham TC et al.; Diffuse-reflectance laser flash photolysis has been used to record transient spectra and decay kinetics of the photodynamic therapy sensitizer disulfonated aluminium phthalocyanine in two murine cancer cell lines, P815 derived from white mouse mast cells, and EL4, a lymphoblast derived from black mouse lymphocytes . In contrast to results with bacterial cells and yeasts, no transient other than the triplet state of the sensitizer was detected, suggesting that unlike the case in microbes, Type I electron-transfer processes play no role in the photodestruction of the murine cells studied.

J Biochem (Tokyo), 2000 Jul, 128(1), 107 - 12
Genome profiling: a realistic solution for genotype-based identification of species; Nishigaki K et al.; Species identification is the basis of Biology and has been carried out based on phenotype . Although some genes, such as that for 16S rRNA, have been used for species confirmation, identification of species based only on genotype has never been done before, although recent whole genome sequencing studies have demonstrated it to be possible in principle . However, it is evidently unrealistic for routine experiments of species identification . This paper clarifies that a very limited amount of information derived from a genome sequence is sufficient for identifying the species . It also proves that Genome Profiling {Nishigaki, K., Amano, N., and Takasawa, T . (1991) Chem . Lett . 1097-1100}, TGGE analysis of random PCR products, can not only fulfill such requirements, but also serve as a universal method to analyze species . Thus, this compact technology can be used in many fields of biology, especially in microbe-related disciplines such as microbial ecology and epidemiology where exact knowledge about all members of a population is essential but previously difficult to obtain . This is the first demonstration that genotype-based identification of species is possible using a simple and uniform protocol for all organisms.

Mol Plant Microbe Interact, 2000 Jul, 13(7), 703 - 14
The endo-beta-1,4-glucanase CelA of Clavibacter michiganensis subsp . michiganensis is a pathogenicity determinant required for induction of bacterial wilt of tomato; Jahr H et al.; The phytopathogenic bacterium Clavibacter michiganensis subsp . michiganensis NCPPB382, which causes bacterial wilt and canker of tomato, harbors two plasmids, pCM1 (27.35 kb) and pCM2 (72 kb), encoding genes involved in virulence (D . Meletzus, A . Bermpohl, J . Dreier, and R . Eichenlaub, 1993, J . Bacteriol . 175:2131-2136; J . Dreier, D . Meletzus, and R . Eichenlaub, 1997, Mol . Plant-Microbe Interact . 10:195-206) . The region of pCM1 carrying the endoglucanase gene celA was mapped by deletion analysis and complementation . RNA hybridization identified a 2.4-knt (kilonucleotide) transcript of the celA structural gene and the transcriptional initiation site was mapped . The celA gene encodes CelA, a protein of 78 kDa (746 amino acids) with similarity to endo-beta-1,4-glucanases of family A1 cellulases . CelA has a three-domain structure with a catalytic domain, a type IIa-like cellulose-binding domain, and a C-terminal domain . We present evidence that CelA plays a major role in pathogenicity, since wilt induction capability is obtained by endoglucanase expression in plasmid-free, nonvirulent strains and by complementation of the CelA- gene-replacement mutant CMM-H4 with the wild-type celA gene.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 643 - 56
Challenge of investigating biologically relevant functions of virulence factors in bacterial pathogens; Moxon R et al.; Recent innovations have increased enormously the opportunities for investigating the molecular basis of bacterial pathogenicity, including the availability of whole-genome sequences, techniques for identifying key virulence genes, and the use of microarrays and proteomics . These methods should provide powerful tools for analysing the patterns of gene expression and function required for investigating host-microbe interactions in vivo . But, the challenge is exacting . Pathogenicity is a complex phenotype and the reductionist approach does not adequately address the eclectic and variable outcomes of host-microbe interactions, including evolutionary dynamics and ecological factors . There are difficulties in distinguishing bacterial 'virulence' factors from the many determinants that are permissive for pathogenicity, for example those promoting general fitness . A further practical problem for some of the major bacterial pathogens is that there are no satisfactory animal models or experimental assays that adequately reflect the infection under investigation . In this review, we give a personal perspective on the challenge of characterizing how bacterial pathogens behave in vivo and discuss some of the methods that might be most relevant for understanding the molecular basis of the diseases for which they are responsible . Despite the powerful genomic, molecular, cellular and structural technologies available to us, we are still struggling to come to grips with the question of 'What is a pathogen?'

Ann Rheum Dis, 2000 Jul, 59(7), 565 - 70
Effect of a three month course of ciprofloxacin on the outcome of reactive arthritis; Yli-Kerttula T et al.; BACKGROUND: Treatment of reactive arthritis (ReA) with antibiotics has so far remained controversial . Eradication of the causative microbe appears logical, but short term antibiotic treatment has no beneficial effect on the outcome of ReA . OBJECTIVE: To evaluate the effect of a three month course of ciprofloxacin on ReA . METHODS: In a randomised, double blind, placebo controlled trial, between December 1992 and February 1996, 71 patients with acute ReA triggered by a gastrointestinal or a urogenital infection were randomly assigned to receive ciprofloxacin 500 mg or placebo twice daily for three months . Patients were assessed at study entry, at 6 weeks, 3 months, 6 months, and 12 months . Sixty two patients were valid for the efficacy analysis . The primary outcome measures were erythrocyte sedimentation rate, number of swollen joints, patients self assessment, and complete recovery . RESULTS: Adverse events were mostly mild and occurred in both treatment groups . There were no statistically significant differences in any of the primary or secondary efficacy variables between the study groups at baseline or during the 12 month follow up . All primary outcome measures indicated that the condition of the patients improved during the study . CONCLUSION: Both groups tended to recover . Ciprofloxacin, given as a three month course, had no advantage over placebo treatment.

Chem Biol, 2000 Jun, 7(6), R127 - 32
Resisting resistance: new chemical strategies for battling superbugs; Wright GD; As microbes become increasingly resistant to antibiotics, and in many cases to several drugs simultaneously, the search is on to find new therapies . One method to combat resistance is to use inhibitors of resistance mechanisms to potentiate existing antibiotics . Recent efforts are encouraging and highlight the importance of research at the chemistry-microbiology interface in developing new approaches to tackle resistance.

QJM, 2000 Jun, 93(6), 375 - 83
Respiratory infection and coronary heart disease: progression of a paradigm; Grimes DS et al.; We have developed a previously published paradigm concerning causation of coronary heart disease, based on the probability that the fundamental cause is a microbe, probably Chlamydia pneumoniae, and that the progress of the disease is influenced by number of accelerating and inhibiting factors . We propose that cigarette smoking acts via respiratory infection, this itself being influenced by immunocompetence resulting from sunlight exposure . We also propose an immuno-enhancing effect of oestrogen and an anti-inflammatory effect of statin therapy . In respect of the geographical variation of coronary heart disease, we emphasize that this must be viewed as part of the bigger picture of a high mortality from all causes in countries of North-west Europe that have a particularly low level of sunlight exposure . Finally, we draw attention to the Albanian, French, Italian, Northern Ireland and Scottish paradoxes which should lead to a major review of the conventional wisdom concerning the aetiogenesis of coronary heart disease.

Geriatrics, 2000 Jun, 55(6), 44 - 9; quiz 50
H pylori infection . Review of the guideline for diagnosis and treatment; Anderson J et al.; Helicobacter pylori infection is prevalent among persons over age 60, is strongly associated with peptic and duodenal ulcer, and is caused by a microbe classified as a carcinogen . These factors combine to make the primary care physician key to proper diagnosis and treatment of H pylori infection . In 1998, the American College of Gastroenterology published an evidence-based guideline for the management of this infection . The guideline produced several fundamental recommendations that help clarify the management process: asymptomatic persons should not undergo testing, but testing should be performed on certain persons, testing should only be done if the intention is to treat; the choice of test is governed by the need for endoscopy; and several triple-therapy regimens are effective for eradication.

Planta, 2000 May, 210(6), 932 - 7
Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor; Kadota A et al.; The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens . When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities . When the E-vector was parallel to the cross-wall (i.e . perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e . parallel to the protonemal axis) . When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used . Red light of 0.1-18 W m-2 and blue light of 0.01-85.5 W m-2 induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m-2 or higher and by blue light of 285 W m-2 . The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment . These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells . Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d.

Can J Microbiol, 2000 May, 46(5), 490 - 3
Evidence for the occurrence of nucleotide-activated oligosaccharides in Saccharomyces cerevisiae; Sprenger S et al.; Recently, nucleotide-activated oligosaccharides have been found to be involved in the biosynthesis of certain glycoconjugates in archaeal and bacterial procaryotes . This paper describes the isolation and partial chemical characterization of nucleotide-activated oligosaccharides from the eucaryotic microbe Saccharomyces cerevisiae . We purified four different nucleotide-activated oligosaccharides from cell extracts of Saccharomyces cerevisiae . Three of the oligosaccharides were UDP, and one was TDP-activated . D-Glucose was the only carbohydrate constituent, except for one oligosaccharide, which also contained glucosamine . The chain length varied between two and four sugar residues.

Nucleic Acids Res, 2000 Jun 15, 28(12), 2353 - 62
Bacterial cryptochrome and photolyase: characterization of two photolyase-like genes of Synechocystis sp . PCC6803; Hitomi K et al.; Photolyase is a DNA repair enzyme that reverses UV-induced photoproducts in DNA in a light-dependent manner . Recently, photolyase homologs were identified in higher eukaryotes . These homologs, termed crypto-chromes, function as blue light photoreceptors or regulators of circadian rhythm . In contrast, most bacteria have only a single photolyase or photolyase-like gene . Unlike other microbes, the chromosome of the cyanobacterium SYNECHOCYSTIS: sp . PCC6803 contains two ORFs (slr0854 and sll1629) with high similarities to photolyases . We have characterized both genes . The slr0854 gene product exhibited specific, light-dependent repair activity for a cyclo-butane pyrimidine dimer (CPD), whereas the sll1629 gene product lacks measurable affinity for DNA in vitro . Disruption of either slr0854 or sll1629 had little or no effect on the growth rate of the cyanobacterium . A mutant lacking the slr0854 gene showed severe UV sensitivity, in contrast to a mutant lacking sll1629 . Phylogenetic analysis showed that sll1629 is more closely related to the cryptochromes than photolyases . We conclude that sll1629 is a bacterial cryptochrome . To our knowledge, this is the first description of a bacterial cryptochrome.

Yeast, 2000 Jun 15, 16(8), 773 - 84
Budding yeast as a model organism for population genetics; Zeyl C; Population genetics is a highly theoretical field in which many models and theories of broad significance have received little experimental testing . Microbes are well-suited for empirical population genetics since populations of almost any size may be studied genetically, and because many have easily controlled life cycles . Saccharomyces cerevisiae is almost ideal for such studies as the growing body of knowledge and techniques that have made it the best characterized eukaryote genome also allow the experimental manipulation and analysis of its population genetics . In experiments to date, the evolution of laboratory yeast populations has been observed for up to 1000 generations . In several cases, adaptation has occurred by gene duplications . The interaction between mutation, selection and genetic drift at varying population sizes is a major area of theoretical study in which yeast experiments can provide particularly valuable data . Conflicts between gene-level and among-cell selection, and co-evolution between genes within a genome, are additional topics in which a population genetics perspective may be particularly helpful . The growing field of genomics is increasingly complementary with that of population genetics . The characterization of the yeast genome presents unprecedented opportunities for the detailed study of evolutionary and population genetics . Conversely, the redundancy of the yeast genome means that, for many open reading frames, deletion has only a quantitative effect that is most readily observed in competitions with a wild-type strain .

J Geol, 2000 Jul, 108(4), 487 - 496
Major Element, REE, and Other Trace Element Behavior in Amphibolite Weathering under Semiarid Conditions in Southern India; Sharma A et al.; A body of komatiitic amphibolite, an enclave within the Archean high-grade orthogneisses in southern India, shows mild chemical weathering under semiarid conditions . Along fractures, chemical weathering has advanced (Chemical Index of Alteration &sqbl0;CIA&sqbr0;=53; CIA of fresh rock approximately 26) to the extent that secondary Mg-Fe-Al clay minerals have formed and the rock has turned brownish red, soft, and fine grained . The weathering process has resulted in the mobilization and redistribution of the so-called immobile elements Fe, Al, Ti, and REE effected by the nature of secondary mineral formation (talc vs . aluminous clay minerals) and also possibly by soil microbes . In the initial stages of secondary mineral formation, there is a small loss of Fe, Al, and REE (noticeably Eu) . However, in the fracture zone as well as in the incipiently altered zone, there is significant REE enrichment, probably affected by a different precipitation mechanism . Mobilized REE may have come from a minor alteration of clinopyroxene.

Curr Opin Struct Biol, 2000 Jun, 10(3), 380 - 3
Structural genomics of microbes: an objective; Kim SH; A comparison of the genome sequences of more than 20 microorganisms reveals that a large fraction of the genes have unknown functions . Determining the structures of the proteins coded by these genes may provide additional key information in an effort to uncover the molecular functions of such proteins and new protein fold patterns . Using existing technology, it is possible to obtain a complete sequence complement and a near complete structural complement for a small microbial genome . Such information may provide a comprehensive view of a small organism, which, in turn, can serve as a platform for understanding more complex organisms.

Curr Opin Struct Biol, 2000 Jun, 10(3), 343 - 8
Genome data: what do we learn?
Nierman WC, Eisen JA, Fleischmann RD, Fraser CM.
Genome sequence information has continued to accumulate at a spectacular pace during the past year . Details of the sequence and gene content of human chromosome 22 were published . The sequencing and annotation of the first two Arabidopsis thaliana chromosomes was completed . The sequence of chromosome 3 from Plasmodium falciparum, the second sequenced malaria chromosome, was reported, as was that of chromosome 1 from Leishmania major . The complete genomic sequences of five microbes were reported . Approaches to using data from completely sequenced microbial genomes in phylogenetic studies are being explored, as is the application of microarrays to whole genome expression analysis.

J Microsc, 2000 Jun, 198 ( Pt 3), 182 - 7
Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules; Greulich KO et al.; Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules . Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers . This may generate new interest in these tools for plant biologists . Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries . Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product . In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae . As many plant cells are transparent, it is possible to work inside living, intact cells . New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations . An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research . As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission) . A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools . For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis . Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries . Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen . The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e . a type of single molecule restriction analysis is possible . Finally, mechanical properties of individual DNA molecules can be observed directly.

Mol Microbiol, 2000 May, 36(4), 775 - 83
New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress; Poole RK et al.; Globin-like oxygen-binding proteins occur in bacteria, yeasts and other fungi, and protozoa . The simplest contain protohaem as sole prosthetic group, but show considerable variation in their similarity to the classical animal globins and plant globins . Flavohaemoglobins comprise a haem domain homologous to classical globins and a ferredoxin-NADP+ reductase (FNR)-like domain that converts the globin into an NAD(P)H-oxidizing protein with diverse reductase activities . In Escherichia coli, the prototype flavohaemoglobin (Hmp) is clearly involved in responses to nitric oxide (NO) and nitrosative stress: (i) the structural gene hmp is upregulated by NO and nitrosating agents; (ii) purified Hmp binds NO avidly, but also converts it to nitrate (aerobically) or nitrous oxide (anaerobically); (iii) hmp mutants are hypersensitive to NO and nitrosative stresses . Here, we review recent advances in E . coli and the growing number of microbes in which globins are known, draw particular attention to the essential chemistry of NO and related reactive species and their interactions with globins, and suggest that microbial globins have additional functions unrelated to 'NO' stresses.

Adv Drug Deliv Rev, 1998 May 4, 31(3), 267 - 285
Protein release from alginate matrices; Wee S et al.; There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins . Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent . Alginate, a naturally occurring biopolymer extracted from brown algae (kelp), has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biological agents . Alginate polymers are a family of linear unbranched polysaccharides which contain varying amounts of 1,4'-linked beta-D-mannuronic acid and alpha-L-guluronic acid residues . The residues may vary widely in composition and sequence and are arranged in a pattern of blocks along the chain . Alginate can be ionically crosslinked by the addition of divalent cations in aqueous solution . The relatively mild gelation process has enabled not only proteins, but cells and DNA to be incorporated into alginate matrices with retention of full biological activity . Furthermore, by selection of the type of alginate and coating agent, the pore size, degradation rate, and ultimately release kinetics can be controlled . Gels of different morphologies can be prepared including large block matrices, large beads (>1 mm in diameter) and microbeads (<0.2 mm in diameter) . In situ gelling systems have also been made by the application of alginate to the cornea, or on the surfaces of wounds . Alginate is a bioadhesive polymer which can be advantageous for the site specific delivery to mucosal tissues . All of these properties, in addition to the nonimmunogenicity of alginate, have led to an increased use of this polymer as a protein delivery system . This review will discuss the chemistry of alginate, its gelation mechanisms, and the physical properties of alginate gels . Emphasis will be placed on applications in which biomolecules have been incorporated into and released from alginate systems.

Annu Rev Immunol, 2000, 18, 275 - 308
CD8+ T cell effector mechanisms in resistance to infection; Harty JT et al.; Based on T cell subset depletion studies and the analysis of gene knockout mice, it is evident that CD8(+) T cells contribute to resistance against intracellular infections with certain viral, protozoan, and bacterial pathogens . Although they are known primarily for their capacity to kill infected cells, CD8(+) T cells elaborate a variety of effector mechanisms with the potential to defend against infection . Microbes use multiple strategies to cause infection, and the nature of the pathogenhost interaction may determine which CD8(+) T cell effector mechanisms are required for immunity . In this review, we summarize our current understanding of the effector functions used by CD8(+) T cells in resistance to pathogens . Analyses of mice deficient in perforin and/or Fas demonstrate that cytolysis is critical for immunity against some, but not all, infections and also reveal the contribution of cytolysis to the pathogenesis of disease . The role of CD8(+) T cell-derived cytokines in resistance to infection has been analyzed by systemic treatment with neutralizing antibodies and cytokine gene knockout mice . These studies are complicated by the fact that few, if any, cytokines are uniquely produced by CD8(+) T cells . Thus, the requirement for CD8(+) T cell- derived cytokines in resistance against most pathogens remains to be defined . Finally, recent studies of human CD8(+) T cells reveal the potential for novel effector mechanisms in resistance to infection.

J Photochem Photobiol B, 2000 Feb, 54(2-3), 175 - 84
Chromosomes are target sites for photodynamic therapy as demonstrated by subcellular laser microirradiation; Liang H et al.; The present investigation has been undertaken to examine the possibility that the cell nucleus, and specifically the genetic material, is a target site for photodynamic therapy . PTK2 and Hep-2 cells are pretreated with a medium containing 15 microg/ml (0.09 mM) 5-aminolevulinic acid (ALA) . Individual fluorescence images are recorded for each selected cell using a cooled charge-coupled device (CCD) . A laser microbeam system generating 630 nm is used for subcellular-region irradiation of specific targets: chromosomes, the mitotic spindle, the perispindle region and the peripheral cytoplasm . Nuclei of interphase cells are also irradiated . Data comparing the sensitivities of the different subcellular microirradiation sites in ALA-treated mitotic cells demonstrate that under the irradiation conditions used, the chromosome is the most sensitive subcellular target followed by the perispindle region, the peripheral cytoplasm and spindle, and, lastly, the interphase nucleus.

Nat Biotechnol, 2000 Jun, 18(6), 630 - 4
Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays; Brenner S et al.; We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads . After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2 . Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation . Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes . The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1 . The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

J Clin Neurosci, 1999 Jan, 6(1), 78 - 82
A novel hybrid biological embolic material: autologous fibroblast incorporated collagen (FC) beads; Nishi S et al.; If viable fibroblasts are used as an active ingredient of an embolic material, it is expected that proliferation and extracellular matrix production of fibroblasts incorporated at a vascular lesion will help restore tissues due to endovascular scar formation, resulting in progressive and permanent occlusion . Based on this working principle, we have devised a novel biological embolic material, hybrid fibroblast incorporated collagen (hybrid FC) beads composed of collagen microbeads and autologous fibroblasts harvested from the subcutaneous tissue of the host to be treated . Hybrid FC beads were prepared by culture of fibroblasts harvested from canine subcutaneous tissue on collagen microbeads (diameters ranging from around 100 microm to around 400 microm) . Canine kidneys were embolized with either hybrid FC or cell-free collagen beads via a transarterial route . Histological examination up to 6 months after embolization revealed that, although both embolic materials effectively occluded the target vessels at the time of embolization, intravascular scar formation activity at the embolized sites was much more profound in the case of the hybrid FC beads than in that of the cell-free beads . Proliferation of autologous fibroblasts was verified by the expression of alkaline phosphatase activity of gene-transfected fibroblasts at the site of lodgement of the beads . It is expected that, using the novel hybrid biological embolic material, hybrid FC beads used for vascular lesions such as arteriovenous malformations can be treated more effectively to restore tissues, resulting in minimal recanalization which often occurs when synthetic embolic materials are used .

FEMS Microbiol Lett, 2000 Jun 1, 187(1), 15 - 20
Degradation of benzyl ether bonds of lignin by ruminal microbes; Kajikawa H et al.; We examined microbial activity in the rumen to cleave benzyl ether bonds of lignin model compounds that fluoresced when the bonds were cleaved . 4-Methylumbelliferone veratryl ether dimer was degraded completely within 8 h even in the presence of fungicidal antibiotics, but no significant degradation occurred with bactericidal antibiotics . Degradation of a phenolic beta-O-4 trimer incorporating 4-methylumbelliferone by a benzyl ether linkage was stimulated by ruminal microbes, although its corresponding non-phenolic model compound, 1-(4-ethoxy-3-methoxyphenyl)-1-O-(4-methylumbelliferyl)-2-(2-methoxyp henoxy)-3-propanol, was not degraded . A coniferyl dehydrogenation polymer bearing fluorescent beta-O-4 benzyl ether that contains both phenolic and non-phenolic benzyl ether bonds was partially degraded (about 20%) in 48 h . These results suggest that ruminal microbes decompose benzyl ether linkages of lignin polymers under anaerobic conditions.

J Cell Sci, 2000 Jun, 113 ( Pt 12), 2207 - 19
Immobilized dimers of N-cadherin-Fc chimera mimic cadherin-mediated cell contact formation: contribution of both outside-in and inside-out signals; Lambert M et al.; Cell adhesion receptors of the cadherin family are involved in various developmental processes, affecting cell adhesion and migration, and also cell proliferation and differentiation . In order to dissect the molecular mechanisms of cadherin-based cell-cell adhesion and subsequent signal transduction to the cytoskeleton and/or cytoplasm leading to adapted cell responses, we developed an approach allowing us to mimic and control cadherin activation . We produced a dimeric N-cadherin-Fc chimera (Ncad-Fc) which retains structural and functional properties of cadherins, including glycosylation, Ca(2+)-dependent trypsin sensitivity and the ability to mediate Ca(2+)-dependent self-aggregation of covered microbeads . Beads covered with either Ncad-Fc or anti-N-cadherin antibodies specifically bound to N-cadherin expressing cells . Both types of beads induced the recruitment of N-cadherin, beta-catenin, alpha-catenin and p120, by lateral mobilization of preexisting cell membrane complexes . Furthermore, cadherin clustering elicited by Ncad-Fc beads triggered local accumulations of tyrosine phosphorylated proteins, a recruitment and redistribution of actin filaments, as well as local membrane remodeling . These results support a model where the adhesion of cadherin ectodomains is followed by clustering of cadherin/catenin complexes allowing signal transduction affecting both cytoskeletal reorganization and cytoplasmic signal mobilization (outside-in signaling) . Interestingly, bead-cell binding was altered by agents promoting microfilament and microtubule depolymerization or tyrosine phosphorylation, indicating a possible regulation of the adhesive properties of the extracellular domain of N-cadherin by intracellular factors (inside-out signaling).

Prenat Diagn, 2000 May, 20(5), 407 - 10
The use of transferrin for enrichment of fetal cells from maternal blood; Serlachius M et al.; Iron loaded transferrin (holotransferrin) was used for enrichment of fetal cells from peripheral blood of pregnant women . Cord blood samples were used to evaluate enrichment efficacy of single and double MACS separations . Blood samples were obtained from 10 pregnant women prior to chorion villus sampling (CVS) . Erythroblasts and other mononuclear cells were isolated by triple-density gradient centrifugation . Fetal cells were further enriched by positive magnetic sorting (VarioMACS) using biotinylated transferrin and streptavidin conjugated to magnetic microbeads . The isolated cells were analysed with dual-colour in situ hybridization (FISH) with X- and Y-chromosome specific probes . Male fetuses were correctly identified in three out of four (75%) pregnancies and female fetuses in six out of six (100%) pregnancies . By using transferrin instead of antibodies to the transferrin receptor to label and enrich fetal cells, we believe that unspecific binding attributed to immunological labelling can be minimized . The results indicate that transferrin may be an alternative to antibodies to transferrin receptor for separation of fetal cells from maternal blood .

Microbes Infect, 2000 Apr, 2(4), 381 - 9
The role of interleukin 15 in mounting an immune response against microbial infections; Yoshikai Y et al.; Interleukin (IL)-15 is a recently described cytokine that resembles IL-2 in its biological activities, stimulating natural killer cells, T cells and B cells to proliferate, secrete cytokines, and exhibit increased cytotoxicities or produce antibody . IL-15 also exerts unique functions such as stimulation of phagocytes, maintenance of mast cells and migration of activated/memory T cells . IL-15 is involved in protection against infections with a variety of microbes through not only activating innate immunity but also mounting adaptive immunity.

Plant Mol Biol, 2000 Mar, 42(5), 689 - 701
A peanut seed lipoxygenase responsive to Aspergillus colonization; Burow GB et al.; Several lines of evidence have indicated that lipoxygenase enzymes (LOX) and their products, especially 9S- and 13S-hydroperoxy fatty acids, could play a role in the Aspergillus/seed interaction . Both hydroperoxides exhibit sporogenic effects on Aspergillus spp . (Calvo, A., Hinze, L., Gardner, H.W . and Keller, N.P . 1999 . Appl . Environ . Microbiol . 65: 3668-3673) and differentially modulate aflatoxin pathway gene transcription (Burow, G.B., Nesbitt, T.C., Dunlap, J . and Keller, N.P . 1997 . Mol . Plant-Microbe Interact . 10: 380-387) . To examine the role of seed LOXs at the molecular level, a peanut (Arachis hypogaea L.) seed gene, PnLOX1, was cloned and characterized . Analysis of nucleotide sequence suggests that PnLOX1 encodes a predicted 98 kDa protein highly similar in sequence and biochemical properties to soybean LOX2 . The full-length PnLOX1 cDNA was subcloned into an expression vector to determine the type(s) of hydroperoxide products the enzyme produces . Analysis of the oxidation products of PnLOX1 revealed that it produced a mixture of 30% 9S-HPODE (9S-hydroperoxy-10E, 12Z-octadecadienoic acid) and 70% 13S-HPODE (13S-hydroperoxy-9Z, 11E-octadecadienoic acid) at pH 7 . PnLOX1 is an organ-specific gene which is constitutively expressed in immature cotyledons but is highly induced by methyl jasmonate, wounding and Aspergillus infections in mature cotyledons . Examination of HPODE production in infected cotyledons suggests PnLOX1 expression may lead to an increase in 9S-HPODE in the seed.

Immunol Rev, 2000 Apr, 174, 35 - 46
The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses; Seki S et al.; The liver remains a hematopoietic organ after birth and can produce all leukocyte lineages from resident hematopoietic stem cells . Hepatocytes produce acute phase proteins and complement in bacterial infections . Liver Kupffer cells are activated by various bacterial stimuli, including bacterial lipopolysaccharide (LPS) and bacterial superantigens, and produce interleukin (IL)-12 . IL-12 and other monokines (IL- 18 etc.) produced by Kupffer cells activate liver natural killer (NK) cells and NK1.1 Ag+ T cells to produce interferon-gamma and thereby acquire cytotoxicity against tumors and microbe-infected cells . These liver leukocytes and the T helper 1 immune responses induced by them thus play a crucial role in the first line of defense against bacterial infections and hematogenous tumor metastases . However, if this defense system is inadequately activated, shock associated with multiple organ failure takes place . Activated liver NK1.1 Ag+ T cells and NK cells also cause hepatocyte injury . NK1.1 Ag+ T cells and another T-cell subset with an intermediate T-cell receptor, CD 122+CD8+ T cells, can develop independently of thymic epithelial cells . Liver NK cells and NK1.1 Ag+ T cells physiologically develop in situ from their precursors, presumably due to bacterial antigens brought from the intestine via the portal vein . NK cells activated by bacterial superantigens or LPS are also probably involved in the vascular endothelial injury in Kawasaki disease.

Biochim Biophys Acta, 1999 Dec 10, 1489(1), 107 - 16
Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides; Krieg AM; Immune stimulation has been widely recognized as an undesirable side effect of certain antisense oligodeoxynucleotides (ODN) which can interfere with their therapeutic application . It is now clear that these dose-dependent immune stimulatory effects primarily result from the presence of an unmethylated CpG dinucleotide in particular base contexts ('CpG motif) . The sequence-specific immune activation is not just an experimental artifact, but is actually a highly evolved immune defense mechanism whose actual 'goal' is the detection of microbial nucleic acids . In contrast to vertebrate DNA, in which CpG dinucleotides are 'suppressed' and are highly methylated, microbial genomes do not generally feature CpG suppression or methylation {1} . Immune effector cells such as B cells, macrophages, dendritic cells, and natural killer cells appear to have evolved pattern recognition receptors (PRR) that by binding the microbe-restricted structure of CpG motifs, trigger protective immune responses . Although the specific immune activation appears to have a variety of potential therapeutic applications, it is generally undesirable in antisense ODN . Immune stimulation may be avoided in antisense oligos by the selection of CpG-free target sequences, by the use of ODN backbones that do not support immune stimulation, or by selective modifications of the cytosine in any CpG dinucleotides.

Trends Ecol Evol, 2000 Jun, 15(6), 238 - 243
The role of polyphenols in terrestrial ecosystem nutrient cycling; Hattenschwiler S et al.; Interspecific variation in polyphenol production by plants has been interpreted in terms of defense against herbivores . Several recent lines of evidence suggest that polyphenols also influence the pools and fluxes of inorganic and organic soil nutrients . Such effects could have far-ranging consequences for nutrient competition among and between plants and microbes, and for ecosystem nutrient cycling and retention . The significance of polyphenols for nutrient cycling and plant productivity is still uncertain, but it could provide an alternative or complementary explanation for the variability in polyphenol production by plants.

Microbes Infect, 1999 Mar, 1(3), 187 - 95
Gammadelta T cells and Mycobacterium tuberculosis; Boom WH; Since the first descriptions of mycobacterial reactivity for gammadelta T cells in 1989, studies of gammadelta T-cell responses to M . tuberculosis in humans and animal models have increased our understanding of the complex role(s) of this T-cell subset not only in the immune response to M . tuberculosis, but also to microbial pathogens in general . Although CD4+ T cells remain the dominant and critical T-cell subset in protection against M . tuberculosis, gammadelta T cells appear to have an important complementary role, which may be primarily expressed in and around maturing granulomas . This is a difficult area to study in humans . Gammadelta T cells are potent sources of IFN-gamma and competent cytotoxic effector cells, but differ from CD4+ T cells in the antigens they recognize and the manner in which M . tuberculosis-infected macrophages process and present antigens to these two subsets . One of the most fascinating features of Vgamma9/Vgamma2+ gammadelta T cells is their responsiveness to non-peptidic molecules . Solving the mechanism(s) of antigen recognition and presentation of these molecules to gammadelta T cells should help determine whether gammadelta T cells are responding to universal 'supernatigen'-like motifs expressed by a broad range of microbes or in fact discriminate among a diversity of peptidic and nonpeptidic microbial antigens . Enhanced understanding of the function of and antigen recognition by Vgamma9+/Vgamma2+ T cells is not only important for immunity to M . tuberculosis but also for T-cell responses to microbial pathogens in general.

Alcohol Clin Exp Res, 2000 Apr, 24(4), 553 - 9
Ethanol and murine interleukin (IL)-12 production; Mason CM et al.; BACKGROUND: Interleukin (IL)-12 is a cytokine with protean effects against bacterial and intracellular pathogens . Induction of IL-12 at the time of infection has salient effects on elimination of various microbes . This work describes the effect of exposure to ethanol on lipopolysaccharide (LPS)-induced production of IL-12 in mice and whether ethanol-induced increases in IL-10 mediates these changes in IL-12 production . METHODS: BALB/c mice were pretreated with ethanol and then challenged with LPS either intravenously (iv) or intratracheally (it), and blood and lung production of IL-12 (p70 and p40 components) and serum IL-10 were assayed . Splenic and lung mRNA for IL-12 p35 and p40 components was determined . RESULTS: Ethanol pretreatment suppressed LPS-induced IL-12 p70 and p40 protein production in blood and lung . In spleen and lung, p40 mRNA was induced to a greater extent than p35 mRNA, and there was greater suppression of p40 mRNA compared with p35 mRNA in ethanol-treated animals . Ethanol up-regulated the production of IL-10, and pretreatment of these animals with a polyclonal anti-IL-10 antibody resulted in significant increases in IL-12 p70 and p40 levels, but not completely to those of control animals . CONCLUSIONS: Ethanol suppresses the production of murine IL-12 in response to LPS in blood and lung, with both the p70 and the p40 components affected . This suppression is accompanied by reductions of p40 mRNA in both spleen and lung . IL-10 may play a role in ethanol-induced suppression of IL-12, as neutralization of IL-10 partially attenuated the suppression of IL-12.

J Appl Microbiol, 2000 May, 88(5), 907 - 13
Disinfection kinetics: a new hypothesis and model for the tailing of log-survivor/time curves; Lambert RJ et al.; A new hypothesis for the understanding of chemical disinfection, which we have termed the Intrinsic Quenching hypothesis, is presented . This mechanistic treatment of disinfection kinetics is based on the hypothesis that the biocide concentration may not be in vast excess over the microbes, as is normally assumed . A mathematical model was developed and found to be useful in describing the observed kinetics of several disinfectants . The model suggested that the reason for the observation of non-linear, log-survivor curves was due to the ability of the microbes, in clean, soil-free conditions, to intrinsically quench the bulk concentration of biocide.

Anaesthesia, 2000 May, 55(5), 458 - 65
The bacterial and viral filtration performance of breathing system filters; Wilkes AR et al.; The bacterial and viral filtration performance of 12 breathing system filters was determined using test methods specified in the draft European standard for breathing system filters, BS EN 13328-1 . All the filters were of two types, either pleated hydrophobic or electrostatic, and these two types differed in their filtration performance . The filtration performance is expressed in terms of the microbial penetration value, defined as the number of microbes passing through the filter per 10 million microbes in the challenge . The geometric mean (95% confidence limits) microbial penetration value was 1.0 (0.5, 3.5) and 2390 (617, 10 000) for the pleated hydrophobic and electrostatic filters, respectively, for the bacterial challenge, and 87 (48, 212) and 32 600 (10 900, 84 900), respectively, for the viral challenge . In general, there was little change in the microbial penetration values following 24 h simulated use . It is concluded that results from the tests specified in the draft standard will allow comparisons to be made between different manufacturers' products enabling an informed choice to be made.

Microb Ecol, 2000 Jan, 39(1), 41 - 48
Stability in Natural Bacterial Communities: II . Plant Resource Allocation Effects on Rhizosphere Diazotroph Assemblage Composition; Piceno YM et al.; Plant rhizospheres are dynamic environments in which microbes compete for resources, particularly plant-derived organic carbon/energy sources . Altering plant productivity changes the availability of carbon/energy resources to rhizosphere microbiota . This limitation is expected to intensify competition for the remaining carbon supply and could cause the loss of poor competitors from the assemblage . We clipped or shaded plots of the salt marsh cord grass, Spartina alterniflora, to shift plant carbon resource allocation from the rhizosphere to the aboveground shoots . We then examined key porewater parameters (pH, salinity, H(2)S, NH(+)(4)), diazotrophic activity (by acetylene reduction assay), and diazotroph assemblage composition after 2 weeks or 8 weeks of treatment . The diazotroph assemblage composition was monitored via the polymerase chain reaction using nifH specific primers followed by denaturing gradient gel electrophoresis (DGGE) analysis . Porewater parameters and acetylene reduction rates did not differ significantly among treatments . The DGGE profiles also were very similar across the control and experimental treatments, indicating that no detectable diazotroph species were displaced from the assemblage . This implies that rhizosphere diazotrophs are able to compete successfully against nondiazotrophs, in spite of the high energy requirements of nitrogen fixation . These results show that the species composition of the diazotroph assemblage in the S . alterniflora rhizosphere is stable in the face of short-duration but potentially high-impact variations in carbon resource availability . </hea

Appl Environ Microbiol, 2000 May, 66(5), 2211 - 5
Simultaneous direct counting of total and specific microbial cells in seawater, using a deep-sea microbe as target; Maruyama A et al.; To rapidly and accurately enumerate total and specific microbes in aquatic samples, fluorescent in situ hybridization was combined with direct counting via direct immobilization of cells on a polymer-coated Nuclepore filter . The technique, named FISH-DC, achieved almost complete recovery of total cells and reproducibility of Psychrobacter pacificensis cells of deep-sea origin (error, </=3%) in a mixed culture and in natural seawater . Target cells immobilized on the filter were also successfully enumerated after stringent 3-cycle hybridization and even after a 16-month preservation at -30 degrees C.

Biol Bull, 2000 Apr, 198(2), 225 - 44
The chemical defense ecology of marine unicellular plankton: constraints, mechanisms, and impacts; Wolfe GV; The activities of unicellular microbes dominate the ecology of the marine environment, but the chemical signals that determine behavioral interactions are poorly known . In particular, chemical signals between microbial predators and prey contribute to food selection or avoidance and to defense, factors that probably affect trophic structure and such large-scale features as algal blooms . Using defense as an example, I consider physical constraints on the transmission of chemical information, and strategies and mechanisms that microbes might use to send chemical signals . Chemical signals in a low Re, viscosity-dominated physical environment are transferred by molecular diffusion and laminar advection, and may be perceived at nanomolar levels or lower . Events that occur on small temporal and physical scales in the "near-field" of prey are likely to play a role in cell-cell interactions . On the basis of cost-benefit optimization and the need for rapid activation, I suggest that microbial defense system strategies might be highly dynamic . These strategies include compartmented and activated reactions, utilizing both pulsed release of dissolved signals and contact-activated signals at the cell surface . Bioluminescence and extrusome discharge are two visible manifestations of rapidly activated microbial defenses that may serve as models for other chemical reactions as yet undetected due to the technical problems of measuring transient chemical gradients around single cells . As an example, I detail an algal dimethylsulfoniopropionate (DMSP) cleavage reaction that appears to deter protozoan feeding and explore it as a possible model for a rapidly activated, short-range chemical defense system . Although the exploration of chemical interactions among planktonic microbes is in its infancy, ecological models from macroorganisms provide useful hints of the complexity likely to be found.

Electrophoresis, 2000 Apr, 21(6), 1178 - 86
The analysis of microbial proteomes: strategies and data exploitation; O'Connor CD et al.; Microbes present special opportunities for proteomic analysis that are not yet available for other types of organisms, due mainly to the relative abundance of information on their genomes, their low levels of functional redundancy and their experimental tractability . They are also being used to develop and validate powerful new experimental approaches that surmount some important current limitations in this field . The review surveys the different proteomic procedures that are available and considers the advantages and disadvantages of different experimental strategies . The ways in which microbiologists - and others - can exploit proteomic data are also discussed.

Int Immunol, 2000 May, 12(5), 691 - 700
Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens; Tani K et al.; Defensins released by neutrophils are able to kill a broad spectrum of microbes . They also induce leukocyte migration in vitro and elicit inflammatory leukocyte responses at s.c . injection sites in mice . In vitro experiments showed that human defensins enhanced concanavalin A-stimulated murine spleen cell proliferation and IFN-gamma production . This led us to examine the effects of human defensins on specific immune responses in vivo . BALB/c mice were immunized with 50 microg of keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide and administered with defensins in aqueous solution . Intraperitoneal administration of defensins significantly increased the production of KLH-specific IgG1, IgG2a and IgG2b antibodies 14 days after immunization . In vitro splenic KLH-specific proliferative responses were higher in mice treated with KLH and defensins than in those treated with KLH alone . Increased IFN-gamma and, to a lesser extent, IL-4 production were also detected in the supernatants of ex vivoKLH-activated spleen cells from mice treated with defensins . Finally, defensins significantly enhanced the antibody response to a syngeneic tumor antigen, lymphoma Ig idiotype and also augmented resistance to tumor challenge . These results indicate that defensins act as potent immune adjuvants by inducing the production of lymphokines, which promote T cell-dependent cellular immunity and antigen-specific Ig production . Thus, defensins appear to function as neutrophil-derived signals that promote adaptive immune responses.

Crit Rev Microbiol, 2000, 26(1), 37 - 57
Molecular techniques for determining microbial diversity and community structure in natural environments; Theron J et al.; The ability to quantify the number and kinds of microorganisms within a community is fundamental to the understanding of the structure and function of an ecosystem . The simple morphology of most microbes provides few clues for their identification and physiological traits are often ambiguous . In addition, many organisms resist cultivation, which is essential to their characterization . Recombinant DNA techniques have provided a means whereby many of the obstacles associated with cultivation and description can be overcome and subsequently has allowed many new insights into the complexity of natural microbial communities . Molecular approaches based on 16S ribosomal RNA (rRNA) sequence analysis allow direct investigation of the community structure, diversity, and phylogeny of microorganisms in almost any environment, while quantification of the individual types of microorganisms or entire microbial communities may be addressed by nucleic acid hybridization techniques . Furthermore, the use of fluorescently labeled population-specific rRNA probes allows microscopic examination of individual cells in complex microbial assemblages as well as their interactions in situ . In this review, we discuss strategies for characterizing microbial communities without the need for cultivation.

J Immunol, 2000 May 1, 164(9), 4742 - 51
Molecular mechanisms of target recognition in an innate immune system: interactions among factor H, C3b, and target in the alternative pathway of human complement; Pangburn MK et al.; In the alternative pathway of complement (APC) factor H is the primary control factor involved in discrimination between potential pathogens . The APC deposits C3b on possible Ags, and the interaction with factor H determines whether the initial C3b activates the APC . Factor H is composed of a linear array of 20 homologous short consensus repeats (SCR) domains with many functional sites . Three of these sites are involved in binding C3b and regulating complement activation; others bind to sialic acid and/or heparin and are responsible for host recognition . Using site-directed mutations we have examined the contributions of each of these sites to target discrimination and to functional activities of factor H . Decay acceleration by SCR1-4 of C3/C5 convertases bound to nonactivators was strongly dependent on SCR domains 11-15 and 16-20 . Loss of these regions caused a 97% loss of activity, with SCR16-20 being the most critical (>90% loss) . On APC activators the pattern of site usage was different and unique on each . On yeast, deletion of the 10 C-terminal domains (SCR11-20) had no effect on specific activity . On rabbit erythrocytes, this deletion caused loss of 75% of the specific activity . An examination of binding affinity to C3b on the four cell types demonstrated that factor H exhibits a unique pattern of SCR involvement on each cell . The results reveal a complex molecular mechanism of discrimination between microbes and host in this ancient innate defense system and help explain the different rates and intensities of APC activation on different biological particles.

Arch Otolaryngol Head Neck Surg, 2000 Apr, 126(4), 522 - 8
Cytokine production by sinus lavage, bronchial lavage, and blood mononuclear cells in chronic rhinosinusitis with or without atopy; Jyonouchi H et al.; BACKGROUND: Chronic sinus inflammation may be determined partly by a balance of proinflammatory and counterregulatory cytokines and other mediators in the sinus . However, their mechanistic roles in chronic rhinosinusitis (CRS) are not well understood . OBJECTIVE: To evaluate production of proinflammatory (interferon gamma {IFN-gamma} and interleukin {IL} 12) and counterregulatory cytokines (IL-10 and IL-4) by sinus lavage (SL), bronchial lavage (BL), and peripheral blood mononuclear (PBMN) cells in patients with CRS . METHODS: We analyzed SL, BL, and PB samples obtained at surgery from 26 patients with CRS . Cytokine production was determined by culturing cells with or without stimuli . The results were evaluated in comparison with other inflammatory variables (cytologic findings, total protein, IgG, and lactose dehydrogenase), bacterial cultures, and clinical features . RESULTS: Production of IFN-gamma by SL cells was variable and did not correlate with other inflammatory variables, microbes grown, IL-10/IL-12p40 production by SL cells, or IFN-gamma production by BL or PBMN cells . Production of IL-4 by lavage cells was undetectable . None of 10 patients with elevated IFN-gamma production (>800 pg/10(6) SL cells with mitogen stimuli) had allergic rhinitis, whereas 12 of 16 patients with low IFN-gamma production (<500 pg/10(6) SL cells) had allergic rhinitis with positive reactivity to common aeoroallergens . There was no significant difference in other variables measured between low and high IFN-gamma production groups . CONCLUSIONS: Elevated IFN-gamma production by SL cells may indicate much less possibility of allergic rhinitis in patients with CRS, but other variables measured did not differ in patients with high or low IFN-gamma production by SL cells.

Parazitologiia, 1999 May-Jun, 33(3), 242 - 50
{The epizootiological role of the population organization of the stock of fleas on the long-tailed suslik in a natural focus of plague in Tuva}; Verzhutskii DB; Spatial location of epizootic events in the Tuva plague focus is determined at a considerable degree by the population structure of the flea Citellophilus tesquorum--the main plague microbe vector . Within the enzootic territory occupied by five populations of the long-tailed ground squirrel (Citellus undulatus) there are six populations of C . tesquorum . Each population of fleas has a corresponding autonomic plague focus . Various conditions for the microbe life activity in these populations are recovered . The circulation of the microbe is closely connected with certain intrapopulation groupings of fleas--the nuclei of populations.

Arch Histol Cytol, 2000 Mar, 63(1), 81 - 9
The presence of specialized epithelial cells on the bronchus-associated lymphoid tissue (BALT) in the mouse; Tango M et al.; The aggregation of lymphoid cells in the bronchial mucosa has been named the bronchus-associated lymphoid tissue (BALT) and investigated in comparison with the gut-associated lymphoid tissue (GALT) . To elucidate precisely the structure and function of the BALT, the present study examined the age-related change in the mouse BALT by light microscopy . We also observed the characteristics of the overlying epithelium, especially the lectin-binding properties of the epithelial cells, by the combined use of light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) . By LM, lymphoid aggregates were not recognizable in the bronchial mucosa of young (8-10 week-old) mice, while they were commonly found at the second to fourth branching portions of the bronchial tree in older (32-40 week-old) mice . The epithelium overlying the lymphoid aggregates of the mature mice often contained a large number of mononuclear cells . Lectin cytochemistry revealed that UEA1 (Ulex europaeus agglutinin 1) positive cells were not only restricted to the overlying epithelium of the BALT in the older mice but also found in a cell group in the mucous epithelium at the branching portions in the young mice . Comparison between the LM and SEM images of the UEA1-stained whole mount specimens clarified the surface morphology of the lectin-stained epithelial cells, showing them to be non-ciliated cells with a large number of short microvillous projections on the apical surface . TEM studies further demonstrated that the UEA1 reaction products appeared on the plasma membrane of the non-ciliated cells which often enfolded lymphocytes in the old mice . Latex microbeads, which were administrated intratracheally, were selectively taken up by the UEA1-positive cells of the BALT . These results indicate that the mouse BALT has specialized epithelial cells similar to the UEA1 positive M cells in the GALT and probably functions as a part of the mucosal immune system . This study also showed the possibility that the UEA1 positive cells appear in the mucous epithelium before the formation of the BALT.

Biotechniques, 2000 Apr, 28(4), 676 - 8, 680, 681
Heteroduplex resolution using T7 endonuclease I in microbial community analyses; Lowell JL et al.; Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities . This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques . Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts . Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA . Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I . Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning . These samples were compared with replicates that did not receive the T7 endonuclease I treatment . Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones . Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened . The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.

Infect Immun, 2000 May, 68(5), 2728 - 34
Heterologous expression of Trypanosoma cruzi trans-sialidase in Leishmania major enhances virulence; Belen Carrillo M et al.; Earlier studies showed that mice primed for a few hours with the trans-sialidase (TS) of Trypanosoma cruzi, the agent of Chagas' disease, become highly susceptible to trypanosomal infection . These studies suggest that TS affects parasite virulence independent of antigenic stimulation . Potentially, TS could enhance or reduce the virulence of heterologous microbes depending on the mechanism of TS action and on the type of immune response elicited by the particular parasite . We tested this hypothesis by expressing heterologous TS in Leishmania major, a protozoan parasite that causes cutaneous leishmaniasis and lacks TS and the TS product alpha2-3-linked sialic acid . Leishmania cells transfected with a T . cruzi TS expression construct made high levels of active enzyme, which was present in the promastigotes and shed into the extracellular milieu . TS expression did not affect L . major binding to and entry into cultured macrophages or its tropism for macrophage infection in vivo . However, TS-expressing L . major exhibited elevated virulence in BALB/c mice, as determined by lesion progression, parasite numbers, and macro- and microscopic examination of cutaneous lesions . Several genetic tests proved that the enhanced virulence was directly attributable to TS expression . The results are consistent with TS functioning to sabotage the mouse immune system to confer a growth advantage on T . cruzi and transgenic L . major . These data suggest that heterologous expression of T . cruzi virulence factors in Leishmania may provide a new approach for dissecting their function in vivo.

Infect Immun, 2000 May, 68(5), 2449 - 56
Potentiality of interleukin-18 as a useful reagent for treatment and prevention of Leishmania major infection; Ohkusu K et al.; Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in natural killer cell activation and the T helper 1 (Th1) cell response, particularly in collaboration with IL-12 . Since Th1 cells play a pivotal role in the host defense against infection with intracellular microbes, such as Leishmania major, we investigated whether IL-18 is critically involved in protection against L . major infection by activation of Th1 cells . We administered IL-12 and/or IL-18 daily to L . major-susceptible BALB/c mice . Neither IL-12 (10 ng/mouse) nor IL-18 (1,000 ng/mouse) induced wound healing, while daily injection of IL-12 and IL-18 during the first week after infection strongly protected the mice from footpad swelling by induction and activation of Th1 cells . Furthermore, these mice acquired protective immunity . We also investigated a protective role of endogenous IL-18 by using anti-IL-18 antibody-treated C3H/HeN mice (an L . major-resistant strain) or IL-18 deficient (IL-18(-/-)) mice with a resistant background (C57BL/6) . We found that in the absence of endogenous IL-18, these mice showed prolonged footpad swelling as well as diminished nitric oxide production . However, daily injection of IL-18 into IL-18(-/-) mice corrected their deficiencies, suggesting that these mice have Th1 cells that produce gamma interferon (IFN-gamma) in response to IL-18 . Indeed, these mice had normal levels of Th1 cells . Thus, IL-18 is not responsible for inducing Th1 cells but participates in host resistance by its action in stimulating Th1 cells to produce IFN-gamma . Our results also indicate the high potentiality of IL-18 as a useful reagent for treatment as well as prevention against reinfection.

J Biol Chem, 2000 Jun 23, 275(25), 18611 - 4
Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2; Huffman DL et al.; The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans . A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles . Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner . The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain . Instead, Atx1 allows rapid metal transfer to its partner . This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions . Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates . These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners . In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.

J Anim Sci, 2000 Mar, 78(3), 504 - 14
Effects of supplemental soybean oil level on in vitro digestion and performance of prepubertal beef heifers; Whitney MB et al.; In vitro digestion and growth studies were conducted to evaluate the effects of level of soybean oil inclusion in forage-based diets . In Exp . 1, diets were bromegrass hay (H), bromegrass hay and corn-soybean meal supplement (C), C with 3% added soybean oil (O3), and C with 6% added soybean (O6) . Diets containing supplements were formulated to be isonitrogenous and isocaloric . Treatment means were compared using a single-degree-of-freedom contrast (H vs C, O3, and O6) and orthogonal polynomial contrasts within diets C, O3, and O6 . Diet H had the lowest (P = .0003) IVDMD and a linear decline (P = .0001) in IVDMD was observed from C to O6, but 24-h IVDMD disappearance was greatest (P = .001; quadratic) for O3 . Total VFA increased from C to O3 and then decreased from O3 to O6 (quadratic; P = .001), and acetate:propionate ratio decreased linearly (P = .0001) from C to O6 . Changes in long-chain fatty acids reflected biohydrogenation by ruminal microbes; however, only 18:3 was hydrogenated to the same extent across all diets . In Exp . 2, 36 Angus x Gelbvieh heifers (260.0 +/- 6.0 kg initial BW) were individually fed C, O3, or O6 as mixed rations for 104 d . Diets were formulated to be isonitrogenous and provide ADG of .91 kg . Feed efficiency and ADG was greatest (P < .02; quadratic) for O3 heifers . Serum NEFA increased linearly (P = .02) and serum glucose (P = .02), cholesterol (P = .002), and GH (P = .04) showed a quadratic response to level of dietary soybean oil . Plasma proportions of 16:0, 16:1, 18:0, and 18:1 increased quadratically (P < .03), and 18:2 increased linearly (P < .001) from C to O6 . In Exp . 3, 42 Angus x Gelbvieh heifers (288.7 +/- 6.6 kg initial BW) were divided into six pens (two pens/treatment) in a randomized complete block designed experiment . Rations were delivered as hay plus a top-dressed supplement (C, O3, or O6) . Heifers fed O3 conceived 10 d earlier (quadratic; P = .06) than heifers fed C and O6 . Other production estimates did not differ (P > or = .10) among dietary treatments . Inclusion of soybean oil at 3% of a forage-based diet increased total VFA, many blood metabolites, ADG, and feed efficiency, and it decreased time to conception . Adding soybean oil as 3% of a forage-based diet is an acceptable feeding strategy for developing beef heifers.

Microbes Infect, 2000 Mar, 2(3), 305 - 11
The role of scavenger receptors in the innate immune system; Gough PJ et al.; Akey aspect of the innate immune system is the ability to discriminate between self and infectious nonself . This is achieved through pattern recognition receptors which directly recognise molecular epitopes expressed by microbes . Scavenger receptors (SRs) have been studied primarily due to their ability to bind and internalise modified lipoproteins, suggesting an important role in foam cell formation and the pathogenesis of atherosclerosis . However, the ability of some SRs to function as pattern recognition receptors through their binding of a wide variety of pathogens indicates a potential role in host defence . This review will detail our current understanding of the function of SRs in innate immunity, and in the initiation of aquired immune responses.

Microbes Infect, 2000 Mar, 2(3), 279 - 88
Macrophage lectins in host defence; Linehan SA et al.; Macrophage lectins contribute to host defence by a variety of mechanisms . The best characterised, mannose receptor (MR) and complement receptor three (CR3), are both able to mediate phagocytosis of pathogenic microbes and induce intracellular killing mechanisms . The regulation of the effector functions induced via MR is complex, and may involve both host and microbial factors . Therefore, MR is likely to play a dynamic role in the response to infection; it may act as a classical pattern recognition receptor in phagocytosis, whereas other poorly characterised factors may make a more decisive contribution to its function in physiologic settings . In contrast, the lectin site of CR3 appears to lack host-derived ligands and may be a true pattern recognition receptor . Further studies are required to evaluate the roles of other macrophage lectins in recognition of and responses to microbes.

J Immunol Methods, 2000 Apr 21, 238(1-2), 133 - 41
The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions; Werther K et al.; A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells . In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1 . The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker . The anti-epithelial monoclonal mouse antibody Ber-EP4 was used as the primary capture antibody . In order to permit phenotyping of the isolated carcinoma cells the magnetic beads were removed from the carcinoma cells by DN'ase digestion of the DNA linker between the magnetic bead and the secondary antibody . In an ex vivo model system an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor) . No positive stained cells were found in control experiments with PBMCs without carcinoma cells . Despite a relatively low recovery, the described method will be valuable for the detection of carcinoma cells in cytospin preparations with subsequent phenotyping of the cells for expression of surface antigens . Depending on the chosen antibodies, the method may be useful for the isolation and characterisation of other cell types in various cell suspensions.

Development, 2000 May, 127(9), 1953 - 60
Laser-induced gene expression in specific cells of transgenic zebrafish; Halloran MC et al.; Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters . These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development . Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful . Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter . At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature . Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them . The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells . By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos . As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

J Eukaryot Microbiol, 2000 Mar-Apr, 47(2), 164 - 6
Use of micromanipulation and "feeder layers" to clone the oyster pathogen Perkinsus marinus; Bushek D et al.; Genetic and biochemical characterization of microbes often requires the use of clonal cultures . A method to clone the oyster parasite Perkinsus marinus is described . Individual cells are isolated via micromanipulation and maintained above an actively proliferating "feeder layer" of P . marinus on a 0.45-microm membrane . Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate . The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.

Plant Cell Physiol, 2000 Jan, 41(1), 84 - 93
Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation; Kagawa T et al.; Chloroplast relocation in mesophyll cells of Arabidopsis thaliana was observed microscopically and analyzed by microbeam irradiation . Chloroplasts located along the anticlinal walls in dark-adapted cells . When part of a cell was irradiated with a microbeam of high fluence rate blue light (B) simultaneously with background red light (R) on the whole cell, the chloroplasts moved towards the B-irradiated area, but did not enter the beam . The background R illumination activated cytoplasmic motility as well as chloroplast movement . Without R illumination, there was little chloroplast relocation . In light-adapted cells in which the chloroplasts were spread over the cell surface perpendicular to the incident light, R-illumination had the same effect . Under background R, the chloroplasts moved out of the area irradiated with a B microbeam of 8 or 30 W m(-2) (avoidance response), but chloroplasts outside the beam moved towards the area irradiated with the B microbeam (accumulation response) . These results suggest that the signals for accumulation and avoidance responses were generated in a single cell by high fluence rate B . cry1cry2, npq1 and nph1 mutants showed B-induced chloroplast relocation . Both the accumulation and avoidance responses were observed in all the mutants, although in the nph1 mutant, the sensitivity of accumulation movement was slightly lower than that of the wild type . We discuss the possible photoreceptor for B-induced chloroplast relocation.

Ceska Gynekol, 2000 Jan, 65(1), 33 - 7
{Levels of peripheral circulating nucleated erythrocytes in pregnant women for noninvasive prenatal diagnosis}; Hromadnikova I et al.; OBJECTIVE: Enrichment of nucleated red blood cells (NRBCs) from maternal blood for non-invasive prenatal diagnosis . DESIGN: Pilot study . SETTING: 2nd Clinic of Paediatrics, University Hospital Motol, Prague, Czech Republic . METHODS: Mononuclear cells were isolated from 13-28 ml of peripheral maternal blood between 13 and 37 weeks of gestation . Leukocytes from maternal peripheral blood were depleted from mononuclear cells by treatment with anti-CD14 and anti-CD45 microbeads and high-gradient magnetic cell separation (MACS) on VarioMACS . NRBCs were sorted from CD14-/CD45- fraction by positive selection using anti-CD71 microbeads on MiniMACS . All sorting steps were analysed by three-colour cytometric analysis with FACScan flow cytometer . RESULTS: In 68 out of 78 pregnant woman (87%) NRBCs were found in range 2 x 10(5) - 1.02 x 10(6) . NRBC were enriched with an average enrichment rate of 138-fold ranging from 4-526 fold . In our cohort of pregnant woman the number of isolated NRBCs was individual . We identified NRBCs from the 13th week of gestation . CONCLUSION: The aim of the study is to establish and standardise the method of enrichment of NRBCs from maternal blood samples and verify the applicability of this alternative source for non-invasive prenatal diagnosis.

J Zoo Wildl Med, 1999 Dec, 30(4), 564 - 72
Pulmonary silicosis in three North American river otters (Lutra canadensis); Suedmeyer WK et al.; Three adult female North American river otters (Lutra canadensis) demonstrated severe anesthetic complications shortly after being immobilized for dental procedures . Two of the animals died shortly after anesthesia, and the third otter died 2 mo after immobilization . All three animals were diagnosed with pulmonary silicosis on the basis of histopathology, polarized light microscopy, and mass spectrometry . One animal also had primary pulmonary bronchoalveolar carcinoma . Significant concurrent disease was not found in other organs . Analysis of the lung tissue by laser microbe mass spectrometry confirmed the presence of an aluminum silicate in the lung tissue associated with insulation material used in nest box construction.

Lang Speech, 1998 Jul-Dec, 41 ( Pt 3-4), 399 - 417
Articulatory correlates of prosodic control: emotion and emphasis; Erickson D et al.; This study examines mandibular correlates of prosodic control in nonread dialog exchanges, in which the subject is asked to repeat the same correction of one digit in a three-digit sequence consisting of "five" or "nine" followed by "Pine Street." Articulatory and acoustic data were collected for four speakers of American English at the X-ray Microbeam Facilities at the University of Wisconsin . Jaw opening was measured as vertical jaw position at the time of maximum opening . Middle digits perceived by independent listeners as emphasized generally show jaw opening which is larger than the average jaw opening for the utterances in which they occur . As the speaker repeatedly makes the same correction, not only does jaw opening increase significantly on the corrected digit but also the overall amount of jaw opening on all digits in the corrected exchanges increases . Independent separate perception tests show that listeners also perceive the speakers' answers to be more irritated as the speaker repeats the same correction . The findings suggest a local and global use of the jaw opening gesture to produce both linguistic or paralinguistic and extralinguistic information, that is, word emphasis and the emotional tenor of the dialog itself.

Plast Reconstr Surg, 2000 Apr, 105(4), 1375 - 81
Effect of hyperbaric oxygen on neutrophil CD18 expression; Larson JL et al.; Previous work has shown that treatment with hyperbaric oxygen significantly reduces neutrophil adhesion to postcapillary venules in a rat microcirculation model of ischemia-reperfusion injury . The mechanism of this process is unknown . The purpose of this study was to evaluate the effect of hyperbaric oxygen on neutrophil CD18 adhesion sites by flow cytometry in an animal model of ischemia-reperfusion injury . The gracilis muscle flap was raised in three groups of male Wistar rats: (1) a sham group (n = 25), (2) a group that underwent 4 hours of ischemia (n = 25), and (3) a group that underwent 4 hours of ischemia and received hyperbaric oxygen (100% 02, 2.5 atmospheres absolute, during the last 90 minutes of ischemia) (n = 25) . Samples from one subgroup of each group (n = 5) were divided into two portions, and one portion was stimulated with phorbol-12 myristate 13-acetate (PMA) . Samples from another subgroup of each group (n = 5) were treated in the same manner, and a flap flush was added at the end of reperfusion to determine the number of CD18 adhesion sites on adherent neutrophils remaining in the flap . Venous blood was drawn 10 minutes after the operation, at 5 minutes of reperfusion, and at 90 minutes of reperfusion . Hematocrit and white blood cell count were measured . Samples were analyzed by flow cytometry, and the antibody binding capacity was assessed using microbead standards and linear regression (antibody binding capacity was expressed as the mean number of sites per cell +/- SEM) . Microbeads were used to align the flow cytometer and to provide external and internal standards . Ischemia-reperfusion injury increased the expression of CD18 by neutrophils (p < 0.05) . Expression of CD18 was not decreased by hyperbaric oxygen treatment . Stimulation with PMA increased the expression of CD18 in all groups (p < 0.01) . These results suggest that ischemia-reperfusion injury does increase the expression of CD18 by neutrophils . Hyperbaric oxygen, as administered in this experiment, did not prevent the increase in CD18 expression.

Bioinformatics, 1999 Nov, 15(11), 900 - 8
An automated comparative analysis of 17 complete microbial genomes; Bansal AK; MOTIVATION: As sequenced genomes become larger and sequencing becomes faster, there is a need to develop accurate automated genome comparison techniques and databases to facilitate derivation of genome functionality; identification of enzymes, putative operons and metabolic pathways; and to derive phylogenetic classification of microbes . RESULTS: This paper extends an automated pair-wise genome comparison technique (Bansal et al., Math . Model . Sci . Comput., 9, 1-23, 1998, Bansal and Bork, in First International Workshop of Declarative Languages, Springer, pp . 275-289, 1999) used to identify orthologs and gene groups to derive orthologous genes in a group of genomes and to identify genes with conserved functionality . Seventeen microbial genomes archived at ftp://ncbi.nlm.nih.gov/genbank/genomes have been compared using the automated technique . Data related to orthologs, gene groups, gene duplication, gene fusion, orthologs with conserved functionality, and genes specifically orthologous to Escherichia coli and pathogens has been presented and analyzed . AVAILABILITY: A prototype database is available at ftp:/// orthos.html . The software is free for academic research under an academic license . The detailed database for every microbial genome in NCBI is commercially available through intellibio software and consultancy corporation (Web site: . html) . CONTACT: arvind@mcs.kent.edu.

J Microbiol Methods, 2000 Mar, 40(1), 11 - 7
Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns; Deng MQ et al.; We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum . Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C . parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column . Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained . The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts . IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples . Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.

Neurogenetics, 1997 May, 1(1), 21 - 8
Multiple sclerosis: a polygenic disease involving epistatic interactions, germline rearrangements and environmental effects; Karpuj MV et al.; Several regions of the human genome are associated with susceptibility to multiple sclerosis (MS) . We review studies of linkage of MS to germline genes using microsatellites . A modest effect on susceptibility was seen with markers in the vicinity of 6p21 (HLA) and 17q22 . The influence of epistatic interactions between these genes is considered . The impact of genetic rearrangements of certain germline genes on susceptibility to MS is described . Analysis of TCR gene rearrangements has established some of the target antigens of the immune response in MS . Environmental influences on MS are described with particular attention given to how microbes might trigger demyelinating disease.

FEMS Microbiol Lett, 2000 Apr 1, 185(1), 9 - 16
Exploration of deep intraterrestrial microbial life: current perspectives; Pedersen K; Intraterrestrial life has been found at depths of several thousand metres in deep sub-sea floor sediments and in the basement crust beneath the sediments . It has also been found at up to 2800-m depth in continental sedimentary rocks, 5300-m depth in igneous rock aquifers and in fluid inclusions in ancient salt deposits from salt mines . The biomass of these intraterrestrial organisms may be equal to the total weight of all marine and terrestrial plants . The intraterrestrial microbes generally seem to be active at very low but significant rates and several investigations indicate chemolithoautotrophs to form a chemosynthetic base . Hydrogen, methane and carbon dioxide gases are continuously generated in the interior of our planet and probably constitute sustainable sources of carbon and energy for deep intraterrestrial biosphere ecosystems . Several prospective research areas are foreseen to focus on the importance of microbial communities for metabolic processes such as anaerobic utilisation of hydrocarbons and anaerobic methane oxidation.

Artif Cells Blood Substit Immobil Biotechnol, 2000 Mar, 28(2), 155 - 71
Attachment of 3T3 and MDBK cells onto poly(EGDMA/HEMA) based microbeads and their biologically modified forms; Ayhan H et al.; Poly(EGDMA/HEMA) based microbeads were prepared by suspension polymerization . A comonomer, i.e., 2-hydroxyethylmethacrylate (HEMA) was included in the recipe in order to have functional hydroxyl groups on the microbead surfaces . Toluene was used in the polymerization formulations to introduce porosity into the matrix . Hydroxyl groups were first oxidized with NaIO4, and then two biological molecules, namely collagen and fibronectin were immobilized by using glutaraldehyde . A spacer-arm, i.e., hexamethylene diamine, was also used in some cases . More protein molecules were immobilized onto more swellable microbeads using spacer-arm . Higher amounts of collagen were immobilized, more than fibronectin immobilization . Attachment of two cell lines (i.e., 3T3 and MDBK cell lines) on these microbeads with a wide variety of surface properties was studied in vitro culture media . Attachments of both cells even onto the plain microbeads were significant . More cells did attach to more swellable microbeads . Introducing both fibronectin and collagen onto the microbeads caused significant increase in the cell attachment . More cells attached to the microbeads carrying fibronectin covalently attached onto the microbeads through the spacer-arm molecules . Fibronectine was better than collagen for high attachment values . The mathematical model proposed successfully simulated attachment kinetics.

Int J Radiat Oncol Biol Phys, 2000 Mar 15, 46(5), 1247 - 57
Studies of physiology and the morphology of the cat LGN following proton irradiation; Reder CS et al.; PURPOSE: We have examined the effects of proton irradiation on the histologic and receptive field properties of thalamic relay cells in the cat visual system . The cat lateral geniculate nucleus (LGN) is a large structure with well-defined anatomical boundaries, and well-described afferent, efferent, and receptive field properties . METHODS AND MATERIALS: A 1.0-mm proton microbeam was used on the cat LGN to determine short-term (3 months) and long-term (9 months) receptive field effects of irradiation on LGN relay cells . The doses used were 16-, 40-, and 60-gray (Gy) . RESULTS: Following irradiation, abnormalities in receptive field organization were found in 40- and 60-Gy short-term animals, and in all of the long-term animals . The abnormalities included "silent" areas of the LGN where a visual response could not be evoked and other regions that had unusually large or small compound receptive fields . Histologic analysis failed to identify cellular necrosis or vascular damage in the irradiated LGN, but revealed a disruption in retinal afferents to areas of the LGN . CONCLUSIONS: These results indicate that microbeam proton irradiation can disrupt cellular function in the absence of obvious cellular necrosis . Moreover, the area and extent of this disruption increased with time, having larger affect with longer post-irradiation periods.

Circulation, 2000 Mar 21, 101(11), 1319 - 23
Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta; Kandabashi T et al.; BACKGROUND: We recently demonstrated that the Rho-kinase-mediated pathway plays an important role for coronary artery spasm in our porcine model with interleukin-1beta (IL-1beta) . In this study, we examined whether or not Rho-kinase is upregulated at the spastic site and if so, how it induces vascular smooth muscle hypercontraction . METHODS AND RESULTS: Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta-bound microbeads . Two weeks after the operation, as reported previously, intracoronary serotonin repeatedly induced coronary hypercontractions at the IL-1beta-treated site both in vivo and in vitro, which were markedly inhibited by Y-27632, one of the specific inhibitors of Rho-kinase . Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of Rho-kinase mRNA was significantly increased in the spastic compared with the control segment . Western blot analysis showed that during the serotonin-induced contractions, the extent of phosphorylation of the myosin-binding subunit of myosin phosphatase (MBS), one of the major substrates of Rho-kinase, was significantly greater in the spastic than in the control segment and that the increase in MBS phosphorylations was also markedly inhibited by Y-27632 . There was a highly significant correlation between the extent of MBS phosphorylations and that of contractions . CONCLUSIONS: These results indicate that Rho-kinase is upregulated at the spastic site and plays a key role in inducing vascular smooth muscle hypercontraction by inhibiting myosin phosphatase through the phosphorylation of MBS in our porcine model.

Ann Ist Super Sanita, 1999, 35(3), 411 - 9
Significance of bacteria in the mucilage phenomenon in the northern Adriatic Sea; Azam F et al.; Episodes of massive mucilage formation in the northern Adriatic Sea have been recorded for over a century but their cause is still a matter of conjecture and debate . It is generally thought that mucilage forms due to copious polysaccharide exudation by phosphorus limited algae . In this paper we develop the thesis that bacteria play major roles in mucilage formation . We argue that mucilage is largely produced as a consequence of bacteria-organic matter interactions and bacterial capsular polysaccharide synthesis . Ectohydrolytic enzymes of bacteria are critical in producing long-lived polysaccharides . Further, bacteria cause efficient P regeneration, particularly intensely in microscale features e.g . phycospheres, detritus and aggregates . Bacteria thus help sustain high rates of primary production despite vanishingly low levels of phosphorus in the bulk phase seawater . We integrate these roles of bacteria into a conceptual model which emphasizes microscale interactions of microbes within a seawater gel matrix as the basis for a mechanistic understanding of the accumulation of long-lived polysaccharide to form mucilage.

J Infect Dis, 2000 Mar, 181(3), 1027 - 33
Segmented filamentous bacteria prevent colonization of enteropathogenic Escherichia coli O103 in rabbits; Heczko U et al.; Despite their distribution in the intestines of many mammals, including man, segmented filamentous bacteria (SFB) have not been found in rabbits, nor has any function been identified for these uncultivable microbes . New Zealand White rabbits were infected with rabbit enteropathogenic Escherichia coli O103 (REPEC O103) derivatives, followed up clinically, and randomly killed 1-4 days after inoculation . Intestinal tissue samples were examined by electron and light microscopy to search for SFB and to evaluate REPEC O103 colonization . Twelve of 21 rabbits showed SFB colonization on ileal absorptive villi . The presence of SFB was correlated with lack of REPEC 0103 ileal colonization (P<.01) and disease . Rabbits without SFB were always colonized by this pathogen . SFB appear to inhibit intestinal colonization by REPEC O103 and thus protect against REPEC 0103 disease . SFB colonization in rabbits is also described for the first time.

Immunol Rev, 2000 Feb, 173, 89 - 97
Innate immune recognition: mechanisms and pathways; Medzhitov R et al.; The innate immune system is an evolutionarily ancient form of host defense found in most multicellular organisms . Inducible responses of the innate immune system are triggered upon pathogen recognition by a set of pattern recognition receptors . These receptors recognize conserved molecular patterns shared by large groups of microorganisms . Recognition of these patterns allows the innate immune system not only to detect the presence of an infectious microbe, but also to determine the type of the infecting pathogen . Pattern recognition receptors activate conserved host defense signaling pathways that control the expression of a variety of immune response genes.

IEEE Trans Inf Technol Biomed, 1998 Dec, 2(4), 282 - 91
A system for medical consultation and education using multimodal human/machine communication; Akay M et al.; Recent developments in networking and computing have enabled collaborative biomedical engineering research by geographically separated participants . One of the most promising goals is to use these technologies to extend human intellectual capabilities in medical decision making . These emerging technologies are poised to drastically reduce healthcare cost by providing service at remote locations . This also increases diagnosis capacity since information is made available to experts at any location . In this paper, we propose a novel application of a recently developed interactive and distributed system in medical consultation and education . Our approach builds on the notion that interactive and distributive capabilities of the system are crucial for medical consultation and education . The presented application uses a multiuser, collaborative environment with multimodal human/machine communication in the dimensions of sight, sound, and touch . The experimental setup, consisting of two user stations, and the multimodal interfaces, including sight (eye-tracking), sound (automatic speech), and touch (microbeam pen), were tested and evaluated . The system uses a collaborative workspace as a common visualization space . Users communicate with the application through a fusion agent by eye-tracking, speech, and microbeam pen . The audio/video teleconferencing is also included to help the radiologists to communicate with each other simultaneously while they are working on the mammograms . The system used in this study has three software agents: a fusion agent, a conversational agent, and an analytic agent . The fusion agent interprets multimodal commands by integrating the multimodal inputs . The conversational agent answers the user's questions and detects human-related or semantic errors and notifies the user about the results of the image analysis . The analytic agent enhances the digitized images using the wavelet denoising algorithm if requested by the user . To show how well the system performs in practice, we used the system for medical consultation on mammograms . Results also show that the relevant information about the region of interest (ROI) of the mammograms chosen by the users is extracted automatically and used to enhance the mammograms.

Dev Comp Immunol, 2000 Mar-Apr, 24(2-3), 85 - 101
Mannan-binding lectin (MBL) in chickens: molecular and functional aspects; Laursen SB et al.; Mannan-binding lectin (MBL) is a serum collectin (i.e . mosaic protein with collagenous and lectin domains) involved in the innate immune defence against various microbes . In vitro studies indicate that MBL exerts its function by binding to the microbial surface through its carbohydrate recognition domains followed by direct opsonization or complement activation via the MBL associated serine proteases MASP-1 and MASP-2 . In Aves (i.e . chickens), as in man, only one MBL form has been found, while traditional laboratory animals (i.e . mouse and rat) have two MBL forms in serum . MBL has been extensively studied in mammals but recently also in Aves . This review summarizes the present knowledge of MBL in chickens and compares it to the situation in mammals.

Br J Surg, 2000 Mar, 87(3), 362 - 73
Extending the indications for curative liver resection by portal vein embolization
Seymour K, Charnley RM, Rose J, Baudouin CJ, Manas DM.
AIMS: The aim of ipsilateral portal vein embolization is to induce hypertrophy of normal tissue when resection of a cancerous portion of the liver is contraindicated only by the volume of liver that would remain following surgery . This study reports its use in primary and metastatic liver tumours . METHODS: Eight patients with inoperable liver tumours (three women and five men of median age 68 . 5 years; three colorectal hepatic metastases, two cholangiocarcinomas and three hepatocellular cancers) were selected for portal vein embolization . Selected portal branches were occluded distally with microbeads and proximally with coils . Liver volumes were determined by magnetic resonance imaging before embolization and again before surgery, 6-8 weeks later . RESULTS: Embolization was performed successfully in seven patients by the percutaneous-transhepatic route; one further patient required an open cannulation of the inferior mesenteric vein . Management was altered in six patients, who proceeded to 'curative' surgery . The projected remaining (predominantly left lobe) liver volumes increased significantly from a median of 350 to 550 ml (P < 0.05, Wilcoxon matched pairs test) . Two patients had disease progression such that surgery was no longer indicated . One patient, whose disease progressed, had the left portal branch occluded unintentionally by a misplaced coil that was successfully retrieved, although the left portal branch remained occluded . CONCLUSIONS: Portal vein embolization produced significant hypertrophy of the normal liver and extended the option of 'curative' surgery to six of the eight patients in whom it was attempted . It appears to be equally effective for primary and metastatic liver tumours in selected patients.

Biochemistry (Mosc), 2000 Feb, 65(2), 164 - 70
Aminopeptidase PC from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica; Rudenskaya GN et al.; Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150 . The enzyme is a homodimer with a molecular mass 220 kD (110 x 2) . Aminopeptidase PC has pI = 4.1 . It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40 degrees C; in the presence of Ca2+ the enzyme is stable at pH 5.5-8.0 . Aminopeptidase PC is activated by Ca2+, Mg2+, and Fe2+; it is completely inhibited by EDTA, o-phenanthroline, and bestatin . The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptidase . The aminopeptidase PC can effectively cleave N-terminal Arg and Lys residues as well as Leu, Phe, and Met residues . Km and kcat values for hydrolysis of Leu-pNA were 0.075 mM and 0.19 sec-1 and for hydrolysis of Arg-pNA 0.078 mM and 0.48 sec-1, respectively . D-Amino acid residues cannot be cleaved . Thus, aminopeptidase PC of the Kamchatka crab has a mixed substrate specificity which is characteristic of some microbe aminopeptidases . Its N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.

Ann Oncol, 2000, 11 Suppl 1, 75 - 80
Translocation t(14;18) in healthy individuals: preliminary study of its association with family history and agricultural exposure; Paltiel O et al.; BACKGROUND: The t(14;18) translocation, present in 90% of follicular non-Hodgkin's lymphomas (NHL), has been found to exist in low levels in healthy persons . Its clinical/prognostic significance in healthy populations is unknown, and risk factors for its development have not been determined . Our objectives were to assess the prevalence of t(14;18) in individuals without NHL, comparing residents of agricultural settlements (kibbutzim) with city dwellers, as well as first degree relatives of NHL cases . PATIENTS AND METHODS: Residents of kibbutzim and members of two control groups: 1) Jerusalem residents--randomly selected hospital administrative workers and 2) first degree family members of lymphoma patients were interviewed extensively regarding exposures and had blood drawn for t(14;18) determination . The translocation was detected after B-cell purification of blood samples with CD-19 microbeads (Mini-Macs) using nested PCR . The method detects the translocation in a BCL2 positive cell line after dilutions of up to 1:10(5) with normal peripheral blood lymphocytes . RESULTS: Nineteen of two hundred thirty healthy individuals (8.3%) tested were found to be positive for t(14;18) . No statistically significant differences in the prevalence of t(14;18) were detected among the rural and urban populations . Five of thirty-four (11.9%) family members tested positive for t(14;18) . No age or sex differences between t(14;18) positive and negative individuals were found . No significant association with exposure to specific agricultural or other chemicals was found . CONCLUSIONS: The presence of the t(14;18) translocation in healthy individuals was not associated with agricultural residence in this preliminary study . Whether relatives of patients with NHL are at increased risk will require further study in larger populations . Specific exposures affecting the onset of this translocation have not been ruled out . The significance of this translocation in healthy individuals remains unknown.

Am Fam Physician, 2000 Feb 15, 61(4), 1047 - 52, 1054
Thyroiditis: differential diagnosis and management; Slatosky J et al.; Thyroiditis is a group of inflammatory thyroid disorders . Patients with chronic lymphocytic thyroiditis (also referred to as Hashimoto's thyroiditis) present with hypothyroidism, goiter, or both . Measurement of serum thyroid autoantibodies and thyroglobulin confirms the diagnosis . Subacute granulomatous thyroiditis (sometimes referred to as de Quervain's disease) is a self-limited but painful disorder of the thyroid . Physical examination, elevated erythrocyte sedimentation rate, elevated thyroglobulin level and depressed radioactive iodine uptake (RAIU) confirm the diagnosis . Subacute lymphocytic thyroiditis (silent thyroiditis) is considered autoimmune in origin and commonly occurs in the postpartum period . Symptoms of hyperthyroidism and depressed RAIU predominate . Acute (suppurative) thyroiditis is a rare, infectious thyroid disorder caused by bacteria and other microbes . The rare, invasive fibrous thyroiditis (Riedel's thyroiditis) presents with a slowly enlarging anterior neck mass that is sometimes confused with a malignancy.

Psychosom Med, 2000 Jan-Feb, 62(1), 40 - 9
Salivary MUC5B-mediated adherence (ex vivo) of Helicobacter pylori during acute stress; Bosch JA et al.; OBJECTIVE: Biochemical host defenses at mucosal sites, such as the oral cavity, play a key role in the regulation of microbial ecology and the prevention of infectious disease . These biochemical factors have distinct features, some of which benefit the host and some that benefit bacteria . We investigated the effects of acute stress on the salivary levels of the carbohydrate structure sulfo-Lewis (sulfo-Le), which is linked to the mucosal glycoprotein MUC5B . Sulfo-Le was recently identified as an adhesion molecule for Helicobacter pylori; therefore, we also measured saliva-mediated adherence (ex vivo) of H . pylori . The oral cavity is suspected to be involved in the transmission of H . pylori . METHODS: Saliva was collected from 17 undergraduates before (baseline), during (stress), and after (recovery) exposure to a video showing surgical procedures . In addition, blood pressure, an impedance cardiogram, and an electrocardiogram were recorded . RESULTS: During stressor exposure, participants reported increased state anxiety . In addition, stroke volume increased and heart rate decreased . The stressor induced a strong increase in salivary sulfo-Le concentration (U/ml), sulfo-Le output (U/min), sulfo-Le/total protein ratio (U/mg protein), and saliva-mediated adherence (ex vivo) of H . pylori . As expected, sulfo-Le concentration correlated with the adherence of H . pylori (r = 0.72, p < .05) . It was demonstrated that the observed adherence was induced by MUC5B and that the carbohydrate structure sulfo-Le contributed to this process . CONCLUSIONS: Our study demonstrated a direct link between stress-mediated biochemical changes and altered host-microbe interactions in humans . Increased bacterial adherence may be a contributing factor in the observed relationship between stress and susceptibility to infectious disease.

Biochemistry, 2000 Mar 14, 39(10), 2499 - 508
Structures of the superoxide reductase from Pyrococcus furiosus in the oxidized and reduced states; Yeh AP et al.; Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide {Jenney, F . E., Jr., Verhagen, M . F . J . M., Cui, X . , and Adams, M . W . W . (1999) Science 286, 306-309} . Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively . SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center . The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans {Coelho, A . V., Matias, P., Fulop, V., Thompson, A., Gonzalez, A., and Carrondo, M . A . (1997) J . Bioinorg . Chem . 2, 680-689} . Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane . In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment . In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules . The iron centers in all four subunits of the reduced structure exhibit pentacoordination . The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

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