Oriented Adhesion of Escherichia coli to Polystyrene Particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

Oriented adhesion of Escherichia coli to polystyrene particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

Mechanism of phagocytosis in dictyostelium discoideum: phagocytosis is mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties

The recognition step in the phagocytotic process of the unicellular amoeba dictyostelium discoideum was examined by analysis of mutants defective in phagocytosis, Reliable and simple assays were developed to measure endocytotic uptake. For pinocytosis, FITC-dextran was found to be a suitable fluid-phase marker; FITC-bacteria, latex beads, and erythrocytes were used as phagocytotic substrates. Ingested material was isolated in one step by centrifuging through highly viscous poly(ethyleneglycol) solutions and was analyzed optically. A selection procedure for isolating mutants defective in phagocytosis was devised using tungsten beads as particulate prey. Nonphagocytosing cells were isolated on the basis of their lower density. Three mutant strains were found exhibiting a clear-cut phenotype directly related to the phagocytotic event. In contrast to the situation in wild-type cells, uptake of E. coli B/r by mutant cells is specifically and competitively inhibited by glucose. Mutant amoeba phagocytose latex beads normally but not protein-coated latex, nonglucosylated bacteria, or erythrocytes. Cohesive properties of mutant cells are altered: they do not form EDTA-sensitive aggregates, and adhesiveness to glass or plastic surfaces is greatly reduced. Based upon these findings, a model for recognition in phagocytosis is proposed: (a) A lectin-type receptor specifically mediates binding of particles containing terminal glucose (E. coli B/r). (b) A second class of "nonspecific" receptors mediate binding of a variety of particles by hydrophobic interaction. Nonspecific binding is affected by mutation in such a way that only strongly hydrophobic (latex) but not more hydrophilic particles (e.g., protein-coated latex, bacteria, erythrocytes) can be phagocytosed by mutant amoebae.     

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.     

Sub-micron particle manipulation in an ultrasonic standing wave: Applications in detection of clinically important biomolecules

Separation of particles from the suspending phase is of interest, among others, to clinical analysts. A system that enables manipulation of sub-micron sized particles in suspensions of analytical scale volume (10–50 l) using a non-cavitating ultrasonic standing wave is described. Particle suspensions, contained in glass capillary tubes of 1–2 mm internal dimension, are treated on the axis of a tubular transducer generating a radial standing wave field at 4.5 MHz. Microparticles (of average diameter range 0.3–10 m) suspended in buffer are concentrated within seconds at preferred regions separated by submillimetre distances. Concentration of suspended latex particles was inhibited in solutions containing protein at levels similar to those occurring in clinical specimens when the suspensions were sonicated in capillaries of circular cross-section. This effect was associated with acoustic streaming of the suspending fluid. Silica microparticles (more dense and less compressible than latex) could be concentrated in the presence of streaming. Latex particles concentrated readily in square cross-section capillaries where no streaming was observed. With sub-micron particles, the geometry of the sample chamber, the suspending phase composition and the size, density and compressibility of the microparticles all influence particle manipulation. The radial standing wave system has been used to enhance agglutination of antibody-coated latex microparticles in the presence of antigen allowing rapid and highly sensitive detection of clinically important biomolecules. The sensitivity of conventional diagnostic tests for microbial antigen has been improved by application of ultrasound and clinical utility has been demonstrated, in particular, for detection of meningitis-causing bacteria.     

Particle size dependent feeding by the rotifer Brachionus plicatilis

Size selective feeding by Brachionus plicatilis was investigated with algae and bacteria (0.3–3.5 µm) and mono-disperse latex beads (0.3–3.0 µm) in short term feeding experiments. B. plicatilis demonstrated maximum clearance rate of particles with diameter 2µm, but particles with diameter down to 0.3 µm were also ingested. The clearance rate of bacteria was 15–55% of that obtained for optimal sized particles (2 µm), and was related to particle size. The relative reduction in retention of particles with diameter < 2 µm was more pronounced for latex beads than for natural food particles, suggesting other mechanisms than size to be important for the particle retention by the rotifer. This is emphasized by the fact that the clearance rates were much lower for latex beads than for natural food particles of comparable size. Efficient retention of bacteria was observed for rotifers in poor physiological condition, i.e. rotifers with low maximum clearance rate. This may reflect a strategy to optimize energy utilization by reducing locomotion costs and increasing energy intake.The results indicate that B. plicatilis has a low to medium ability to feed on bacteria. In natural ecosystems, its importance as a bacterial grazer is of limited importance. At high population densities, such as in live feed cultures, the rotifer may, however, efficiently remove bacteria from the culture.     

Evaluation of the phagocytic activity of peripheral blood neutrophils in patients with hyperthyroidism

In 80 women with hyperthyroidism (40 with diagnosed Graves disease and 40 with hyperactive nodular goiter) the following tests related to the function of peripheral blood neutrophils have been carried out: 1. nitrotetrazolium blue (NRT) reduction test. 2. evaluation of phagocyting activity by using latex particles and living bacteria cells, and 3. determination of immunoglobulin concentration and the C3 component of the complement in blood serum. The following features were found in the patients with hyperthyroidism: 1. the elevated values of the index of spontaneous NBT reduction which return to normal following the treatment with propranolol or metizol lasting 14 days, 2. a decrease in the phagocyting activity of neutrophils occurring with stimulation of phagocytosis by both the latex particles and bacteria cells. 3. the return to normal values of the index of neutrophils phagocyting the latex particles following two-week treatment with propranolol or metizol. It was concluded that in patients with hyperthyroidism the changes in NRT reduction and phagocyting activity of peripheral blood neutrophils return to normal following the two-week treatment of these patients with both propranolol and metizol.     

Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

Retention of ingested latex particles in Peyer's patches of germfree and conventional mice

Conventional and germfree mice ingested a suspension of 2-micron latex particles in drinking water for a 15-day period. Number and distribution of intestinal Peyer's patches did not differ significantly in the two types of mice. Cleared Peyer's patches were compared with regard to size and particle content. The location of particles within Peyer's patch follicles of germfree mice was similar to that of conventional mice, but the latter had significantly larger follicles and greater accumulations of latex particles. Latex concentration varied with patch location. Proximal patches contained the majority of particles in germfree mice, whereas particles were most abundant in distal patches of conventional mice. The results show that particle uptake into Peyer's patches takes place even in the complete absence of bacteria in the gut.     

Use of packed bed bioreactors application to ores bioleaching

Summary The behaviour of glass beads, silicagel and activated carbon particles as bacteria supports for using in backed bed bioreactor has been compared. No important difference was found. Additionally the performance of a bioreactor with glass beads was compared with that of a conventional percolating column in the bioleaching of a copper sulphide ore. Results showed higher copper extraction using the bioreactor.     

Use of a calibrated melamine-formaldehyde latex for the luminescence microscopic study of phagocytosis in a macrophage culture

The possibility has been demonstrated of the use of calibrated melamine-formaldehyde latex for studying phagocytosis as an object not undergoing intracellular digestion. Latex was discovered to be actively phagocytized by macrophages and to exert no toxic action on them. A study was made of the time course of changes in the color and brightness of the fluorescence of the latex phagocytized particles in macrophages intravitally fluorochrominated by acridine orange. These changes were demonstrated to be analogous to those observed previously during phagocytosis of bacteria and other objects. The data obtained demonstrate once more the transition of the fluorescent complex from lysosomes to phagosomes and the lack of the relationship of these changes with intracellular death and digestion of the phagocytized objects. The possibility has been also shown to differentiate between phagocytized particles of latex and those located outside the cells.     

Two distinct mechanisms for stimulation of oxygen-radical production by polymorphonuclear leucocytes.

Oxygen-radical production stimulated from rat polymorphonuclear leucocytes by either unopsonized latex particles (diameter = 1.01 microM) or chemotactic peptide (N-formyl-Met-Leu-Phe) was monitored by using luminol-dependent chemiluminescence. Azide inhibited by more than 80% the luminescence response induced by chemotactic peptide whether added before or after stimulation. However, the luminescence response to latex particles was progressively less susceptible to azide inhibition if the azide was added after the stimulus. Cytochalasin B, which was shown to abolish phagocytosis of the latex beads, also abolished the chemiluminescence response. However, the same cells showed a greatly enhanced response to chemotactic peptide. Cytochalasin B-treated cells secreted approx. 45% of total cellular myeloperoxidase in response to chemotactic peptide, but there was no detectable secretion in response to unopsonized latex particles. Microperoxidase equivalent to 20% of cellular peroxidase activity added to the cells before addition of the stimulus had no effect on the response to latex particles but increased approx. 2-fold the peak rate of chemiluminescence induced by chemotactic peptide. It was concluded that the unopsonized latex particles stimulated oxygen-radical production by the mechanism that involved endocytosis, whereas chemotactic peptide stimulated production by a mechanism that involved exocytosis of myeloperoxidase, the latter mechanism requiring an increase in intracellular free [Ca2+].     

Investigations on cellular defense reactions with Galleria mellonella against Bacillus thuringiensis

In contrast to a great number of foreign particles (bacteria and inorganic materials), cells of some strains of Bacillus thuringiensis are not phagocytosed in the first hours after injection into the hemocoel of Galleria mellonella. This phenomenon is not caused by the production of β-exotoxin or exoenzymes, because heat-killed cells are not phagocytosed and the phagocytosis of latex particles is not prevented by the presence of living B. thuringiensis. The phagocytosis of heat-killed B. thuringiensis subtoxicus can be encouraged by treatment of the cells and by simultaneous injection of latex particles. A factor stimulating the phagocytosis is discussed. It is induced by the injection of phagocytosable latex particles into the hemocoel but not by injection of living or killed B. thuringiensis subtoxicus.     

Coccidioides immitis endospores: Phagocytosis by human cells

Phagocytosis of killed endospores by glass adherent peripheral human mononuclear cells was studied. Phagocytosis continued through 30 minutes of incubation. No difference in rates of ingestion could be detected when cells from coccidioidin-reactive and nonreactive subjects were compared although both groups ingested endospores more avidly than latex particles.     

Effects of the <Emphasis Type="Italic">Papaya meleira virus</Emphasis> on papaya latex structure and composition

Spontaneous latex exudation is the main symptom of papaya sticky (meleira) disease caused by the Papaya meleira virus (PMeV), a double-stranded RNA (dsRNA) virus. This paper describes different effects of PMeV on papaya latex. Latex samples were subjected to different histochemical tests to evaluate their chemical composition. Additionally, the integrity of the latex particles was assessed by transmission and scanning electron microscopy analysis. Biochemical and micro- and macro-element measurements were performed. PMeV dsRNA extraction was performed to evaluate the interaction of the virus with the latex particles. Sticky diseased latex was positive for alkaloid biosynthesis and showed an accumulation of calcium oxalate crystals. PMeV also increased H₂O₂ synthesis within sticky diseased laticifers. The protein, sugar and water levels were altered, probably due to chemical changes. The morphology of the latex particles was further altered; PMeV particles seemed to be bound to the latex particles. The alkaloid and H₂O₂ biosynthesis in the papaya laticifers indicate a papaya defense response against PMeV. However, such efforts failed, as the virus affected the plant latex. The effects described here suggest some advantages of the infection process, including facilitating the movement of the virus within the papaya plant.     

Development and application of thermo-sensitive magnetic immunomicrospheres for antibody purification

Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum.Correspondence to: A. Kondo     

Use of a novel affinity tag selected with a bacterial random peptide library for improving activity retention of glutathione S-transferase adsorbed on a polystyrene surface

Aiming at developing a novel affinity tag for site-specific immobilization of functional proteins onto polystyrene (PS) surfaces, Escherichia coli random peptide display library was screened for dodecapeptides exhibiting a high affinity toward PS plates. The selected peptides were commonly rich in hydrophobic amino acid residues and had two or three basic amino acid residues. Adsorption and desorption experiments for one of the selected peptide named PS1 (KGLRGWREMISL) showed that it was well and irreversibly adsorbed onto PS latex particles. To study its performance as an affinity tag, PS1 was genetically fused to a model enzyme, glutathione S-transferase (GST), in several manners, and the fusion enzymes were compared to the original GST in terms of the adsorption behavior onto the PS latex particles as well as the specific activities before and after the adsorption. The fusion GSTs in solution showed lower specific activities than the original one, and their adsorption behaviors were also altered. In particular, the fusion of PS1 to the N-terminal region of GST resulted in severe losses both in the specific activity and in the adsorptive ability. However, two types of GSTs fused with PS1 at the C-terminal region were well adsorbed onto the PS latex particles, and their specific activities after the adsorption were significantly higher than the original GST adsorbed on the PS latex particles. The fusion of PS1 to the C-terminal region of GST was thus shown to reduce the activity loss upon the adsorption onto the PS latex particles.     

Establishing oil-degrading biofilms on gravel particles and glass plates

A method is described for “artificially” establishing biofilms rich in hydrocarbon degrading bacteria on gravel particles and glass plates. The microbial consortia in the biofilms included in additions, filamentous cyanobacteria, picoplankton and diatoms. Phototrophic microorganisms were pioneer colonizers. Hydrocarbon utilizing bacteria, namely Acinetobacter calcoaceticus and nocardioforms were in part attached to filaments of cyanobacteria. In batch cultures, it was shown that those artificial biofilms had an attenuation effect on crude-oil in contaminated sea water samples. The potential use of these biofilms for preparing trickling filters (gravel particles), and in bioreactors (glass plates) for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed.     

Filter efficiency of commercial face masks in capturing particles and airborne bacteria

The filter efficiency of seven kinds of commercial face mask for particles and airborne bacteria was tested in the wash room of a laboratory animal facility. The filter efficiency of the masks was 19 to 50%, as measured by the weight of particles with diameters below 10 micron, 22 to 71% for particles of the 0.3 micron level, 47 to 90% for the 1 micron level, and 90 to 99.6% for the 5 micron level. The filter efficiency for airborne bacteria was 35 to 81%. Among these even masks tested, glasswool surgery masks, three-sheet synthetic fiber masks with and without charcoal, and 28-sheet gauze masks with glass filter showed generally high efficiency, and single-sheet synthetic fiber masks, 18-sheet of gauze masks and gas masks showed low efficiency.     

Decreased longevity of human diploid cells after incorporation of latex spheres within their cytoplasm

The effects of cytoplasmic incorporation of latex spheres (a cytoplasmic marker) on the growth potential of human diploid cells was examined. After incorporation of latex spheres within their cytoplasm, GM2290 (diploid, Lesch-Nyhan) cells showed a reduced replicative potential. A lower percentage of cells exposed to latex particles incorporated [³H]thymidme during any subsequent 24-h test period when compared with comparable, untreated cells. The overall life expectancy of the cultures treated with spheres was reduced approx. 25%. A similarly treated and examined transformed cell line (HeLa) showed no similar adverse effects after incorporation of latex spheres. The results suggest that latex spheres should be used with caution in experiments on in vitro cellular senescence.