Bacteria biofilm encourages algal immigration onto substrata in lotic systems

I tested the effect of the density of attached bacteria on the amount of algal immigration in the early development of a periphyton community in an artificial stream by manipulating the density of the attached bacteria. Three densities were prepared by regulation of the incubation time. A suspension of algae was added to the stream, and the degree of algal attachment to substrata was compared among the treatments. Algal immigration was proportional to the density of attached bacteria on all substrata (glass, PVC, and slate), although density differed among substrata. Analysis of covariance (dependent variable, amount of attached algae; covariate, bacterial density) showed significant relationship between amounts of attached algae and bacterial densities, but did not show significant differences in the slopes and adjusted means among substrata. When acrylic beads were added with the suspension of attached algae, significant linear correlation was obtained between the amount of attached algae and the amount of acrylic beads on the substrata. Algal immigration was due to non-selective adsorption by attached bacterial biofilms on substrata, although the extent of bacterial colonization and biofilm formation may be affected by the substrata and other environmental factors (e.g., current conditions and water temperature).     

Wet and dry bacterial spore densities determined by buoyant sedimentation

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Wet and dry bacterial spore densities determined by buoyant sedimentation.

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

Understanding enzyme action on immobilised substrates

With increasing interest in automated synthesis and screening protocols, solid supported chemistry and biochemistry are attractive technologies. Studies with surface-immobilised substrates have been carried out to analyse enzyme accessibility, kinetics and thermodynamics. Several interesting new methods have been developed to monitor enzyme action on substrates attached to a solid phase such as polymer beads glass or gold surfaces. These include fluorescence measurements, MALDI-TOF mass spectrometry, and the use of quartz crystal microbalances to measure weight changes of immobilised molecules directly on the surface. Approaches that allow spatial resolution in single beads have also been reported. The ability of enzymes to reach the inside of beads is becoming better characterised and new supports have been developed that allow improved accessibility. The equilibrium position of reactions on the solid surface can be substantially shifted compared with reactions in solution, and this can be usefully exploited using hydrolases in reverse. Research is also starting to tackle the way in which kinetics are modified when the substrates are surface immobilised.     

Isolation and identification of diadenosine 5',5'-P1,P4-tetraphosphate binding proteins using magnetic bio-panning

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.     

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

Chondroitin sulfate-derivatized agarose beads: a new system for studying cation binding to glycosaminoglycans

Chondroitin sulfate (CS) has been covalently attached to aminoethyl-agarose beads in a carbodiimide-catalyzed reaction. In this process, an amide bond is formed between carboxylate groups on the glycosaminoglycan (GAG) and the primary amine groups of the beads. Under optimal conditions, up to 160 micrograms of CS is attached per milligram of beads. CS-agarose beads have been used to study Ca binding to GAGs. The beads are mixed with a solution containing CaCl2 and 45Ca and allowed to sediment under unit gravity. An aliquot of supernatant is then removed and 45Ca activity is determined to quantitate remaining (free) Ca. Using this system, it was shown that CS binds approximately 0.7 Ca/disaccharide unit at saturation. Under the conditions used, the apparent association constant (KA) is approximately 14 mM. In principle, this derivatization protocol may be used to attach any proteoglycan or GAG (except keratan sulfate) to an insoluble support. CS-agarose beads provide a rapid, simple, and relatively artifact-free system for studying cation-GAG interactions.     

Examination of immobilized fungal cells by phase-contrast and scanning electron microscopy

Conidia of Penicillium urticae were immobilized in kappa-carrageenan beads and then shaken, in a growth-supporting medium to yield an in situ grown population of mycelia. The physical stability of these beads and the degree of mycelial growth inside the beads were significantly affected by the concentrations of kappa-carrageenan and locust bean gum (LBG) in the bead matrix and by the porous or nonporous nature of the interior. Thus 16-h-old porous and nonporous beads, prepared from 1.25% kappa-carrageenan, 0.5% LBG, and conidia, possessed a very dense mycelial mass at the surface. Only the porous beads possessed a moderately dense mycelial mass at the centre. The conidia at the centre of nonporous beads either failed to germinate or formed very small germ tubes. When washed, 36-h-old porous beads were repeatedly (i.e., 48 h) transferred into nitrogen-free medium, the density of mycelia at the centre increased to equal that at the surface after three transfers or 8 days. Mycelia at the surface exhibited signs of physical damage, while those in the centre did not. The addition of 100 micrograms/mL of cycloheximide to these replacement cultures was reflected by the distortion of interior mycelia.     

Virus attachment to transparent exopolymeric particles along trophic gradients in the southwestern lagoon of New Caledonia

Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (N) attached to TEP varied as a function of TEP size (d [in micrometers]) according to the formulas N = 100d(1.60) and N = 230d(1.75), respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection.     

Release of prostacyclin,endothelium-derived relaxing factor and endothelin by freshly harvested cells attached to microcarrier beads

Summary Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI₂). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI₂) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permited collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED₅₀ of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI₂ were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.Abbreviations EDRF Endothelium-Derived Relaxing Factor - PGI₂ Prostacyclin - K-H Krebs-Henseleit solution - cyclic GMP cyclic Guanosine Monophosphate - fmoles femtomoles - IB Ibuprofen     

Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation

Non-porous poly(glycidyl methacrylate/ethyleneglycol dimetacrylate) (poly(GMA/EGDMA)) beads were prepared by suspension polymerization. The enzyme (i.e. laccase) was covalently immobilized onto plain and spacer-arm attached poly(GMA/EGDMA) beads. The amount of immobilized enzyme on the plain and spacer-arm attached beads was determined as 5.6 and 4.9 mg/g, respectively. The maximum activity (V(max)) and Michaelis constant (K(m)) of laccase immobilized on the spacer-arm attached beads, were found to be 77.6 U/min and 0.47 mM, respectively. Finally, the immobilized laccase was operated in a batch system, and textile dye Reactive Red 120 was successfully decolorized in the enzyme reactor.     

Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.

Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization.     

Coupling polylysine to glass beads for plasma membrane isolation

Solid glass beads for use in isolating cell membranes were coated with a stable, covalently attached layer of polylysine. The optimal conditions for coating the bead surface were established and the beads were tested by measuring the attachment of human erythrocyte plasma membranes. When compared to other beads, such as those with absorbed polylysine or protamine, none retained red-cell membranes as well as glass beads with covalently linked polylysine.     

Active motor proteins can couple cargo to the ends of growing microtubules

In living cells, dynamic microtubule ends interact with specialized protein complexes located on microtubule targets such as chromosomes and the cell cortex. A significant role in coupling microtubule ends to these complexes has been attributed to motor proteins, which are thought to provide a physical link while at the same time allowing for microtubule growth or shrinkage. In the past, motor-coated beads have been shown to be able to follow the ends of depolymerizing microtubules, in a direction opposite to their natural walking direction. Here we show that beads coated with plus-end-directed motors can also stay attached for several seconds to the ends of growing microtubules. Upon arrival at the microtubule end, fast-moving beads reduce their velocity to the microtubule growth velocity. We show that the tendency to stay attached depends on the initial bead velocity and that the microtubule growth velocity is unaffected by the presence of the bead.     

Mechanical strength of sarcomere structures of skeletal myofibrils studied by submicromanipulation

The mechanical strength of sarcomere structures of skeletal muscle was studied by rupturing single myofibrils of rabbit psoas muscle by submicromanipulation techniques. Microbeads coated with alpha-actinin were attached to the surface of myofibrils immobilized to coverslip. By use of either optical tweezers or atomic force microscope, the attached beads were captured and detached from the myofibrils. During the detachment of the beads, the actin filaments bound specifically to the beads were peeled off from the bulk structures of myofibrils, thus rupturing the peripheral components of the myofibrils bound to the actin filaments. By analyzing the ruptures thus produced in various myofibril preparations, it was found that the sarcomere structure of myofibrils is maintained by numerous molecular components having the mechanical strength sufficient to sustain the contractile force produced by the actomyosin system. The present techniques could be applied to study the mechanical strength of cellular organelles containing actin filaments as their component.     

Improved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum

The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound ¹²⁵I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regards bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads.     

Stimulation of guinea pig lymphocytes by Lens culinaris lectin-A. Stimulation by insolubilized LcL-A and effect of erythrocytes and peritoneal macrophages on stimulation by soluble LcL-A. Presence of a lymphokine in culture fluids of LcL-A-stimulated cells

Guinea pig lymphocytes in culture are mitogenically stimulated by either soluble LcL-A or by LcL-A covalently attached to Sepharose beads. The degree of stimulation by LcL-A-Sepharose is directly dependent upon the density of LcL-A molecules per bead ranging from essentially zero with low density to a better stimulation than by soluble LcL-A with high density. Addition of small numbers of either erythrocytes or peritoneal macrophages to lymphocytes cultured with suboptimal doses of soluble LcL-A increases stimulation significantly. With optimal or supraoptimal doses the potentiation effect disappears. These observations support the proposal that a stimulating lectin presented to stimulatable lymphocytes in high local concentrations is more effective than the same concentration of lectin molecules binding more randomly on the lymphocyte surface. During stimulation of human and guinea pig lymphocytes by LcL-A or LcL-A-Sepharose a soluble lymphokine is released which is either itself a lymphocyte stimulant or one which makes lymphocytes more stimulatable by low doses of LcL-A.     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."