Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.     

Murine models of chronic Pseudomonas aeruginosa lung infection

The animal model of chronic bronchopulmonary infection using agarose beads laden with Pseudomonas aeruginosa is frequently utilized in cystic fibrosis research, though it is challenging to perform it in mice. This paper reports the most successful methods for the creation of this model. Transtracheal insertion of a 22 G 1" over-the-needle intravenous catheter to preferentially inoculate the right mainstem bronchus using tribromoethanol anaesthesia administered i.p. was better for a successful surgical outcome compared, respectively, to the use of a 27 G (1/2)" needle, bilateral inoculation or an anaesthetic cocktail of xylazine, acepromazine and ketamine administered i.p. Bilateral infection was associated with higher mortality, greater weight loss and higher levels of bronchoalveolar cytokine concentration, compared to mice infected primarily in the right lung. Mucoid clinical strain PA M57-15 was preferred since mucoid clinical strain PA 2192 led to comparatively more severe lesions and higher mortality. Using the same operator for a given task reduced the variability inherent in this model, illustrated using outcome measures such as gross lung pathology. The response of mice inoculated with P. aeruginosa-laden agarose beads was characterized by bronchopulmonary inflammation, high production of cytokines, and significant weight loss; whereas the response to infection with free-living bacteria was characterized by pneumonia, lower production of cytokines and weight loss. The use of free P. aeruginosa pre-mixed with sterile agarose beads may be considered as an alternative to the use of P. aeruginosa-laden agarose beads, since the histopathological features were similar, though further characterization is needed to evaluate its utility as an adequate model of cystic fibrosis.     

Sperm binding to an egg model composed of agarose beads

A sperm-binding factor of the eggs of the sea urchin Hemicentrotus pulcherrimus or Anthocidaris crassispina, was coupled to agarose beads. Upon contact with these beads, the spermatozoa exhibited an acrosome reaction and bound to them. No binding occurred to BSA-coupled beads or to beads missing active groups. When coupled beads were pretreated with a sperm factor, which is the probable counterpart to the sperm-binding factor, they could no longer bind sperm. Reciprocal cross-binding between the above two species was not successful, but H. pulcherrimus sperm became capable of binding to A. crassispina beads if they first underwent acrosome reaction in egg water. Sperm of two of seven other echinoderms examined was able to bind to the coupled beads.     

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Novel purification system for 6xHis-tagged proteins by magnetic affinity separation

We have developed a novel nickel-silica matrix for the generation of magnetic beads for metal-ion affinity chromatography. In contrast to magnetic Ni-NTA agarose beads, the novel particle type (SiMAC) consists of a magnetic core and a nickel-silica composite matrix with the nickel ions tightly integrated in the silica. This results in a much higher number of chelating groups compared with Ni-NTA agarose beads. With the SiMAC beads, greatly improved purification of histidine-tagged proteins from crude bacterial extracts was achieved. The yield was at least twice as high as with conventional materials, the method is faster, since the coupling step is omitted and there is no need for handling toxic Ni(2+) salts.     

PREDICTING DESICCATION STRESS IN MICROSCOPIC ORGANISMS THE USE OF AGAROSE BEADS TO DETERMINE EVAPORATION WITHIN AND BETWEEN INTERTIDAL MICROHABITATS1

We describe an easy and inexpensive way to determine whether intertidal microhabitats remain wet during tidal emersion. This new technique uses agarose beads (120 üm diameter when fully hydrated) that shrink in a graded fashion as they dry. The agarose beads allow variability in surface wetness to be gauged over distances of less than 1 mm. Describe this parameter of microclimate is important in order to predict the likelihood and spatial pattern of survival of settled larvae, reproductive propagules, and other microscopic stages in the life histories of organisms growing in intertidal and other water-stressed environments. For the brown seaweed Pelvetia fastigiata (J. Ag.) DeToni (Fucales, Phaeophyta), the use of agarose beads demonstrated that survival of zygotes during tidal emersion was highes at those sites that remain damp. Temperature alone was found to be an unreliable measure of wetness within a single microhabitat (e.g. red algal turf).     

Construction of low melting point agarose emulsion PCR amplification complex gene sequence

Emulsion polymerase chain reaction, an effective amplification, can make millions of templates could be individually amplified within a single tube. Here we constructed and improved a low melting point agarose-emulsion method to promote the specific sequences amplification effectively. Artificial Lactobacillus Plasmid as template was amplified and clear fluorescence images of the agarose beads were obtained. The Real-time PCR data showed that agarose-emulsion PCR clearly indicated that DNA can be amplified in agarose droplets. Overall, our study effectively overcame the difficulty of formation of uniform emulsion droplets, negative effect on recombination of homologous regions of DNA and generation of void emulsion droplets. This method increases the accuracy with amplification, reduces the influence of uncertainties and provides the reliable data for further experiment.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

High-performance molecular sieve chromatography of proteins on agarose columns: the relation between concentration and porosity of the gel

Curves showing the relation between log (molecular weight) and distribution coefficient are presented for proteins subjected to molecular sieve chromatography on crosslinked and non-crosslinked agarose gels of different concentrations. These curves, which facilitate selection of the gel concentration that gives optimal resolution in any particular separation problem, show that the exclusion limit of 5, 9, 12, and 20% agarose gels correspond to protein with molecular weights above 1,000,000, 600,000, 450,000, and 280,000, respectively. Plate numbers have been determined for columns of 20% agarose at different flow rates and bead sizes. Separations of model proteins by high-performance molecular sieve chromatography on agarose beads are shown.     

Coimmobilization of substrate and biocatalyst: A method for bioconversion of poorly soluble substances in water milieu

Coimmobilization of biocatalyst and substrate was studied as a method to increase the conversion rate in systems with substrates of extremely low solubility in water. The system studied was the conversion of hydrocortisone to prednisolone by Arthrobacter simplex. As a matrix for coimmobilization, alginate turned out to be superior to agar and agarose. After the reaction was complete, the beads were solubilized, andthe cells recovered for reuse, by centrifugation, whereas the prednisolone was extracted from the supernatant.     

Short-duration low-direct-current electrical field treatment is a practical tool for considerably reducing counts of gram-negative bacteria entrapped in gel beads

Application of a direct-current electrical field for very short times can serve as a practical nonthermal procedure to reduce or modify the microbial distribution in gel beads. The viability of Escherichia coli and Serratia marcescens entrapped in alginate and agarose beads decreases as the field intensity and duration of electrical field increase.     

Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells

A method is described to prepare total DNA from single cells of dinoflagellates, which can be used for PCR amplification. As model organisms, we used a stock strain of Alexandrium catenella and cells of Dinophysis acuminata harvested from the Atlantic Ocean. Fresh grown cells or cells maintained in different preservatives were tested as sources for DNA preparation. The method used to prepare DNA combines physicochemical and enzymatic procedures on cells embedded in agarose plugs or beads. The agarose pieces containing the DNA were used to perform PCR amplification of a fragment of DNA containing a 5.8S rRNA gene and the flanking internal transcribed spacers (ITS1 and ITS2).     

Protein osmotic pressure and the state of water in frog myoplasm

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.     

Enzyme-based sensor arrays for rapid characterization of complex disaccharide solutions

An enzyme-based sensor array has been developed to detect multiple disaccharides in aqueous solutions. Porous agarose beads, derivatized with enzymes for assaying disaccharides, are localized within wells etched into a silicon chip in a regular 5 x 7 array. Each well is individually addressable and acts as a microanalysis chamber where sample solution passes through the agarose matrix and is exposed to the enzymes. Detection is achieved by observing the increase in absorbance of a quinoneimine dye produced during the reaction. This technique is used to quantify the disaccharides lactose, sucrose, and maltose and the monosaccharide glucose. Preexisting glucose in the sample complicates multicomponent sensing but can be accounted for by including a glucose sensor in the array. This detection strategy is applied to the simultaneous analysis of these sugars in several beverages.     

Properties of cellulase immobilized on agarose gel with spacer

Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated CH-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose.     

Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations.

The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (DG congruent with N0-0.5) to a reptational behavior (DG congruent with N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.     

Involvement of a 40-kDa glycoprotein in food recognition, prey capture, and induction of phagocytosis in the protozoon Actinophrys sol

A 40-kDa glycoprotein (gp40) was identified as a Con A-binding adhesive substance of the heliozoon Actinophrys sol for immobilizing and ingesting prey flagellates. Isolation and partial characterization of gp40 showed that: 1) gp40 is a major Con A-binding protein of Actinophrys with a molecular weight of 40 kDa, and is stored in secretory granules called extrusomes; 2) gp40 was purified by Con A-affinity chromatography, and the N-terminal amino acid sequence was determined as H2N-KVLK-FEDDFDTFDLQ; 3) prey flagellates became adhered to gp40-immobilized agarose beads; 4) phagocytosis of Actinophrys was induced against gp40-immobilized agarose beads; and 5) solubilized gp40 induced exocytosis of extrusomes and cell fusion of heliozoons. These results indicate that gp40 is a multi-functional secretory protein of Actinophrys, which is required in correct targeting of the heliozoon to food organisms as well as in self-recognition.     

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.