Development of Na-CN—κ-carrageenan Microbeads for the Encapsulation of Lipophilic Compounds

The ionotropic gelation of double-layered emulsions composed of sodium caseinate and ??-carrageenan at pH values of 7 and 3.5 was evaluated, in order to obtain potential encapsulation matrices for hydrophobic compounds. The influence of some of the extrusion process variables (nozzle diameter at fluid exit and collecting distance) on the microbead production was studied, as well as the stability of the microbeads. The fluid nozzle diameter showed little influence on the shape of the microbeads, with a slight tendency for a decrease in microbead diameter with increase in fluid nozzle diameter. On the other hand, the collecting distance strongly influenced the microbead shape and they became more spherical (aspect ratio was reduced from ~2.0 to ~1.4) as the collecting distance was increased from 10?cm to 50?cm. The emulsion pH did not affect the aspect ratio of the microbeads, but the diameter was greater for microbeads produced at pH 3.5. This difference was attributed to the kind of interactions occurring between the ??-carrageenan and sodium caseinate at these distinct pH values. The microbeads were highly unstable when dispersed in deionized water, sugar solutions and low salt concentrations, releasing the encapsulated oil. However, no release of oil from the microbeads was observed when they were dispersed in ethanol or potassium chloride solutions with concentrations above 0.75?%, although their shape was modified when dispersed in ethanol. In general, the results obtained demonstrated the viability of the extrusion process to produce biopolymer-based microbeads and the potential application of these systems.     

Electrostatic encapsulation and growth of plant cell cultures in alginate.

The growth of callus tissue from African Violets, encapsulated in alginate using electrostatics, was investigated as well as the mechanism of alginate droplet formation. Alginate microbeads as small as 500 (+/-50) microns in diameter could be produced by electrostatic extrusion directly from a plastic syringe (1900 micron extrusion orifice), in the absence of a needle. Video analysis of the mechanism of electrostatic alginate droplet formation from the syringe showed the development of a Taylor cone-like droplet which extended to form a thin strand that then broke up into droplets. Autoclaving of the alginate/medium solution significantly reduced its viscosity, giving smaller beads. Calculated microbead diameters agreed well with experimental values. Callus tissue from leaf explants was successfully immobilized and cultured using electrostatic extrusion. Tissue immobilized using 4% alginate in medium and cultured on agar grew best, producing a complete plantlet within four months. The long-term aim is to develop an effective method for large production of artificial seeds.     

Immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine

The process of electrostatic extrusion as a method for cell immobilization was investigated that could be used for potential applications in medicine. An attempt was made to assess the effects of cell addition and polymer concentration on the overall entrapment procedure, ie, on each stage of immobilization: polymer-cell suspension rheological characteristics, electrostatic extrusion process, and the process ofgelation. The findings should contribute to a better understanding of polymer-cell interactions, which could be crucial in possible medical treatments. Alginate-yeast was used as a model system for carrier-cells. The electrostatic extrusion was considered as a complex two-phase flow system and the effects of cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under investigated conditions, microbeads 50-600 microm in diameter were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the findings by rheological characterization of the cell-alginate suspensions. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of cell-alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5x10(8) and 1x10(9) cells/mL) did not significantly affect the rheological properties of Na-alginate solution. Lastly, we investigated the kinetics of alginate gelation with respect to the quantity of Ca2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl2 solution. The molar ratio of G units to Ca2+ ions of 3.8:1 provided complete crosslinking, while the increase in alginate concentration resulted in prolonged gelation times but higher strength of the resulting gel. The cell presence decreased the rate of network formation as well as the strength of the obtained Ca-alginate hydrogel.     

Microencapsulation of mammalian cells in a water-insoluble polyacrylate by coextrustion and interfacial precipitation

A process for the microencapsulation of mammalian cells in a commercially available water-insoluble polyacrylate (EUDRAGIT RL) is described, and the effects of process parameters are outlined The polymer dissolved in diethyl phthalate was pumped along the annulus formed from two concentric needles, while the cell suspension was pumped inside the inner needle Droplets of polymer solution containing cells were blown off the end of the needles by a coaxial air stream. The droplets fell into a corn oil-mineral oil curing bath, in which the solvent was removed from the nascent capsule causing the polymer to precipitate around the cell suspension core. Capsules were washed free of oils and solvent in a fractionated plasma that allowed for quantitative transfer of capsules from the oil phase to an aqueous medium. By appropriate adjustment of the coaxial air flow rate, capsule size could be varied from 250-1000 mum, although the most convenient size was found to be 400-700 mum. Adding Ficoll 400 to the cell suspension to match the density of the suspension to the polymer solution resulted in capsules with a well-centered core but did not affect capsule strength. It appeared that increasing the polymer solution concentration or the polymer to the cell flow rate ratio resulted in an increased capsule strength, although differences in capsule size made unequivocal conclusions difficult. These capsules are of potential use as an artificial pancreas for the treatment of diabetes (with pancreatic islets) or for large-scale tissue culture and the production of bioactive products (e.g., with fibroblasts).     

<Emphasis Type="Italic">In vitro</Emphasis> collecting (IVC). I. The effect of collecting method and antimicrobial agents on contamination in temperate and tropical collections

Summary In vitro collecting is the process of initiating tissue cultures in the field. In order for in vitro collecting to be broadly available as a technique for collecting plant germplasm, the levels of contamination in such cultures must be controlled. Two techniques for in vitro collecting were compared: leaf punch and needle collecting. The effectiveness of these methods for collecting leaf and stem tissues from plants at tropical and temperature sites was compared. Stem tissue collected by the needle collecting method gave cultures with an average contamination percentage of 31% and 16%, from the tropical and temperate sites, respectively, while with the leaf punch method, average contamination percentages were 90% and 69%. The effectiveness of antimicrobial agents in reducing contamination in leaf punch cultures was evaluated. Addition of the fungicide benlate and the antibiotics cefotaxime and vancomycin, to the leaf punch collections reduced contamination to an average of 30% in the tropical collections and 35% in the temperate collections. Over 90% of both tropical and temperature species collected in multiple samples of 10 or more had at least one clean sample using this medium. The use of either the leaf punch method in combination with a fungicide and antibiotics or the needle collecting technique yielded a high percentage of clean tissues for study and growth.     

Localized contact formation by erythrocyte membranes: electrostatic effects.

The topology of the contact seam of human erythrocytes adhered by dextran, an uncharged polymer, has been examined. Particular attention has been paid to the influence of electrostatic intermembrane interactions since their magnitude and range can be accurately estimated. Normal cells formed a continuous seam, whereas erythrocytes with pronase-modified glycocalices formed localized contact points on adhesion in 72 kDa dextran in buffered 145 mM NaCl. The dependence of the inter-contact distance lambda on dextran concentration [D] over the range 2-6% w/v, was given by lambda = C[D]-0.62, where C was a constant. The index of [D] was independent of dextran molecular mass over the range 20 to 450 kDa. The inter-contact distance for pronase-pretreated cells in 6% w/v 72 kDa dextran increased from 0.78 to 1.4 microns as [NaCl] was reduced through the range 145 to 90 mM and the suspending phase was maintained at isotonicity by using sorbitol to replace NaCl. The formation and lateral separation of the contact points are discussed from the perspective of linear interfacial instability theory. The theory allows a quantitative explanation for the experimentally observed dependence of inter-contact distance and of disturbance growth rate on change in electrostatic interaction. The results suggest that the dominant wavelength, determining the inter-contact distance, is established on approaching membranes when the layers of cell surface charge are separated by a perpendicular distance of < 14 nm (bilayer separation of 24 nm).     

Zur Abhängigkeit des Putzens von der Konzentration der Zuckerlösung bei der Gemeinen Wespe (Paravespula vulgaris)

Several activities of foraging wasps (Paravespula vulgaris) collecting sucrose solution at an artificial food source are described. The concentration of the solution has an influence on the number of preening movements performed per unit time (= rate of preening). The rate of preening as a function of the concentration is explained in a quantitative manner using a diagram which corresponds to the disinhibition hypothesis for displacement activities.     

Water soluble levan polysaccharide biopolymer electrospun fibers

Electrospinning has been used to prepare nanofibers from diverse biopolymers. Here we report on preparation of fibers by electrospinning of levan (a polysaccharide) from distilled water. A high concentration of levan was required for fiber formation. This suggests that higher concentrations enable the formation of chain entanglements required to maintain the jet strength. In general, fiber diameter decreased with increased voltage, distance between collector plate and needle and decreased pump flow rate. X-ray diffraction of the fibers showed a highly amorphous character in levan formed from solution compared to the levan powder.     

Interactions between Nearest-neighboring Glycosaminoglycan Molecules of Articular Cartilage

The electrostatic interaction effects including the interaction potential, force and torque between the neighboring chondroitin sulfate glycosaminoglycan (CS-GAG) molecular chains in the bottle brush conformation of proteoglycan aggrecan are obtained as the functions of the minimum separation distance and the mutual angle between the molecular chains based on an asymptotic solution of the Poisson-Boltzmann equation that the CS-GAGs satisfy under the normal physiological conditions of articular cartilage. The present study indicates that the electrostatic interactions are not only associated intimately with the separation distance and the mutual angle, which are shown as purely exponential in separation distance and decrease with increasing mutual angle, but also dependent sensitively on the saline concentration in the electrolyte solution within the tissue. Further analysis shows that in the range of the separation distance between two neighboring CS-GAG molecular chains in the bottle brush conformation of the aggrecan in vivo (2~4 nm), if the saline concentration in the electrolyte solution is not less than a value of concentration (~0.1 M), the interactions between the molecular chains will monotonically increase with decreasing the saline concentration, however, if the saline concentration is less than the value of concentration, the relationship between the interactions and the saline concentration will not be simply monotonic. Some present results are in agreement with the existed relevant conclusions.     

Thermodynamics of conformational transition and chain association of ι-carrageenan in aqueous solution: Calorimetric and chirooptical data

Isothermal microcalorimetry, differential scanning calorimetry (DSC), and chirooptical data obtained for ι-carrageenan in NaCl, LiCl, and NaI aqueous solutions are presented. The experiments have been performed as a function of concentration both for the polymer and for the simple salt as a cosolute. The experimental findings consistently show the occurrence of a salt-induced disorder-to-order transition. From microcalorimetric experiments the exothermic enthalpy of transition ΔH_tr is obtained as the difference between the theoretical, purely electrostatic ΔH_el enthalpy change and the actual mixing enthalpy ΔH_mix, measured when a ι-carrageenan salt-free solution at constant polymer concentration is mixed with a 1:1 electrolyte solution of variable concentration. In the case of added NaCl, the absolute values of enthalpy changes |ΔH_tr| are in good agreement with those obtained for the opposite process, at comparable polymer and salt concentrations, from DSC melting curves. The microcalorimetric results show that the negative maximum value of ΔH_tr corresponding to the interaction of Li⁺ counterion with ι-carrageenan polyion results to be significantly lower than the corresponding values obtained for Na⁺ counterion. At variance with the microcalorimetric data, chirooptical results show that the salt-induced disorder-to-order transition, occurring in the 0.02–0.2M salt concentration range, appears to be complete at a concentration of about 0.08–0.1M of the simple ion, irrespective of the polymer concentration and of the nature of added electrolyte. © 1998 John Wiley & Sons, Inc. Biopoly 45: 105–117, 1998     

The role of epidermal turgor in stomatal interactions following a local perturbation in humidity

Humidity in a small area of a Vicia faba L. leaf was perturbed with a flow of dry air from an 80 µm (inside diameter) needle, while the remainder of the leaf was maintained at high and constant humidity. The influence of the needle flow on the humidity at the leaf surface was quantified by using a spatially explicit dewpoint hygrometer to observe condensation patterns. When the dry air from a needle was applied to the leaf, stomata within the influence of the needle opened within the first few minutes of the perturbation, and local epidermal turgor pressure declined within the same time frame. When the needle flow was removed from the leaf, these responses were reversed, but with more variable kinetics. Stomata and epidermal cells outside the influence of the needle flow, which were exposed to a constant and high humidity, showed similar, but smaller, responses when the needle flow was applied to the leaf. Since the opening of these stomata should have had only a small effect on transpiration (because of the high humidity), it is likely that the reduction in epidermal turgor was the cause (rather than the result) of the stomatal opening. The magnitude of the turgor response was only loosely related to the distance from the needle flow up to distances of almost 400 µm. The data support the idea that neighbouring stomata can interact through the influence of transpiration on epidermal turgor.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.     

Impact of Elevated PCO2 on Mass Flow of Reduced Nitrogen in Trees

To analyze the effects of elevated carbon dioxide concentration （PCO2） on the mass flow of reduced nitro- gen （N） in the phloem and xylem of trees, juvenile beech （Fagus sylvatica L.） and spruce （Picea abies （L.） Karst.） were grown in phytotrons and exposed to ambient and elevated PCO2 （plus 687.5 mg/m^3 CO2） for three growing seasons. Elevated PCO2 significantly decreased the mass flow of N from the shoot to roots of beech by significantly reducing the concentration of soluble amino compounds in the phloem, even if the area of conductive phloem of cross-sectional bark tissue was significantly increased, because of less callus deposition in the sieve elements. In spruce, the downward mass flow of reduced N also tended to be decreased, similar to that in beech. Resembling findings in the phloem, N mass flow from roots to shoot in both tree species was significantly diminished owing to significantly reduced concentrations of amino compounds in the xylem and a lower transpiration rate. Therefore, the mass flow of reduced N between shoots and roots of trees was mainly governed by the concentrations of soluble amino compounds in the phloem and xylem in relation to the loading of reduced N in both long-distance transport pathways.     

Molecular dynamics simulations of rubredoxin from Clostridium pasteurianum: Changes in structure and electrostatic potential duringredox reactions

Molecular dynamics simulations of Clostridium pasteurianum rubredoxin in the oxidized and reduced forms have been performed. Good agreement between both forms and crystal data has been obtained (rms deviation of backbone atoms of 1.06 and 1.42 Å, respectively), which was due in part to the use of explicit solvent and counterions. The reduced form exhibits an unexpected structural change: the redox site becomes much more solvent-accessible, so that water enters a channel between the surface and the site, but with little actual structural rearrangement (the rms deviation of backbone atoms between the oxidized and reduced is 0.77 Å). The increase in solvent accessibility is also seen, although to a much lesser extent, between the oxidized and reduced crystal structures of Pyrococcus furiosus rubredoxin, but no high resolution crystal or nuclear magnetic resonance solution data exist for reduced C. pasteurianum rubredoxin. The electrostatic potential at the iron site and fluctuations in the potential, which contribute to both the redox and electron transfer properties, have also been evaluated for both the oxidized and the reduced simulations. These results show that the backbone plays a significant role (62–70 kcall/mol/e) and the polar sidechains contribute relatively little (0–4 kcal/mol/e) to the absolute electrostatic potential at the iron of rubredoxin for both forms. However, both groups contribute significantly to the change in redox state by becoming more polarized and more densely packed around the redox site upon reduction. Furthermore, these results show that the solvent becomes much more polarized in the reduced form than in the oxidized form, even excluding the penetrating water. Finally, the simulation indicates that the contribution of the charged side chains to the electrostatic potential is largely canceled by that of the counterions. © 1995 Wiley-Liss, Inc.     

Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

Effect on Transpiration and Water Uptake by Rapid Changes in the Osmotic Potential of the Nutrient Solution

The sudden changes in the rates of transpiration and water uptake which occurred when the osmotic potential of the nutrient solution surrounding the roots of young wheat plants was rapidly changed were studied. The transpiration was measured by the aid of the microwave hygrometer and the water uptake by a recording poto-meter specially built for this investigation. When the osmotic potential of the nutrient solution was rapidly increased by adding mannitol, there was a temporary transpiration increase. The maximum increase was greater but the total time of the temporary increase shorter when a higher mannitol concentration was used. The quantity of water transpired by the shoots due to the temporary transpiration increase seemed to be fairly constant irrespectively of the mannitol concentration. The water transport to the shoots was immediately reduced when the osmotic potential was rapidly increased. The immediate reduction was greater when a higher mannitol concentration was used. After the immediate reduction the rate of water transport increased without delay. When the osmotic potential of the nutrient solution was rapidly decreased by withdrawing mannitol there was a temporary transpiration decrease, and the water transport to the shoots was immediately increased. After this increase the rate of water transport started to decrease at once. When, however, the mannitol concentration had been 0.30 M or higher, the transpiration rate increased progressively, and the change of the rate of water transport was small. The results indicate that the primary effect of the rapidly changed osmotic potential is localized to the root surface. The rapidly reduced water transport to the shoots after adding mannitol brings about the temporary transpiration increase. The course of events after withdrawing mannitol is just the reverse to that when adding mannitol.     

Growth and ion uptake by maize seedlings on solutions variable in calcium and flow rate

Summary Growth and ion uptake by maize seedlings was studied as functions of calcium concentration and rate of flow of nutrient solutions. With calcium concentrations established at 1, 10, and 100 ppm Ca, all other nutrients being kept at constant concentration, the solutions passed through narrow culture trays at velocities of 2, 8, and 24 liter/day.Fresh and dry weights of seedlings increased as the concentration of calcium increased from 1 to 10 or 100 ppm for any value ofV.Positive response in the plant calcium content was particularly noticed when Ca increased to 100 ppm in the nutrient solution. Phosphorus content of tops decreased with increasing (Ca) in the nutrient solution, while the other elements were more or less unaffected. In the case of roots, phosphorus was generally increased, iron decreased and potassium showed inconsistent trends.Estimates of the uptake efficiency relative to calcium were made by calculating the ratio of the amount of calcium taken up by the entire seedling to the product of calcium concentration and daily flow rate of the solution. It appears that the uptake efficiency diminishes with increasing flow rate. Other instances points to the maintenance of the same uptake efficiency as (Ca) increased with the decreasingV or vice versa. These observations point to the critical nature of environmental factors on the effective contacts between supply and uptake belts that have been adopted as model in this study.     

A new method in growth-electrophysiology: Pressurized intra-organ perfusion

Abstract A new experimental system was devised for the simultaneous measurement of elongation rate and the activity of the spatially separate electrogenic ion pumps of a hypocotyl segment excised from a seedling of Vigna unguiculata L. Walp. under enforced intra-organ perfusion by artificial solutions. The pathway of the perfusion medium was apoplastic space, including xylem vessels as main routes. The elongation rate of the segment was highly dependent on the perfusion pressure applied. It was possible to increase the growth rate under pressurized perfusion by 10-30 times as much as that without perfusion. Elongation rate was also dependent on respiration under perfusion, being retarded reversibly by anoxia a few minutes after the activities of the electrogenic ion pumps were stopped. Perfusion pressure had a little influence on the membrane potential (V_px) below a breakdown level (c. 130 kPa). Perfusion of mannitol or sorbitol solution of appropriate concentration reduced the elongation rate reversibly.