High Dextran Concentrations Research Articles

A novel all-natural (collagen+pectin)/chitosan aqueous two-phase microcapsule with improved anthocyanin loading capacity

Microcapsules based on an aqueous two-phase system (ATPS) have attracted increasing interest due to their all-aqueous biocompatible microenvironment, but the undesirable use of high concentrations of synthetic poly(ethylene glycol) and dextran (Dex) in conventional ATPSs has severely hindered their application in the food industry. In this study, a new type of ATPS microcapsule was developed by taking advantage of the spontaneous phase separation and simultaneous interfacial self-assembly of natural pectin, chitosan, and collagen. Chitosan (1 wt%) and collagen (4 wt%)+pectin (0.5 wt%) were used to form ATPS to replace poly(ethylene glycol) (15∼30 wt%) and Dex (15∼20 wt%) in conventional ATPSs. The (collagen + pectin)/chitosan microcapsules exhibited the plumpest morphology and highest stability at pH 6, and the smoothest interfacial membrane texture was achieved through interfacial self-assembly of pectin, chitosan, and collagen via electrostatic interactions and hydrogen bonds. Notably, anthocyanin could be stabilized by pectin in the inner phase, and the (collagen + pectin)/chitosan microcapsules had an improved anthocyanin encapsulation efficiency and loading capacity of 92.58% and 12.34 g/100 g, respectively, which was attributed to the electrostatic attractions between the flavylium cation form of anthocyanin and anionic pectin. This study yielded a strategy for constructing a new type of food-grade ATPS microcapsule using all-natural polysaccharides and proteins, and provided a novel all-aqueous template for loading hydrophilic bioactive components. • Natural chitosan and collagen + pectin generate a spontaneous aqueous two-phase system. • The novel (collagen + pectin)/chitosan microcapsules developed were stable at pH 6. • Chitosan, pectin, and collagen could be self-assembled at the microcapsule interface. • Anthocyanin could be stabilized by pectin in the inner phase via electrostatic attraction. • (Collagen + pectin)/chitosan microcapsules had excellent anthocyanin loading capacity.

Electrospun PVA–Dextran Nanofibrous Scaffolds for Acceleration of Topical Wound Healing: Nanofiber Optimization, Characterization and In Vitro Assessment

Electrospun polyvinyl alcohol–dextran (PVA–Dex)-based nanofibers (NFs) are explored as a novel class of bioactive injury dressing materials, which have an essential role for topical injury mending. Sodium ampicillin-loaded citric acid-cross-linked PVA–Dex NFs were fabricated by electrospinner for wound recuperating purposes. Results revealed that PVA (10%)–dextran (10%) cross-linked with 5% citric acid (CA) was chosen as an optimized condition for obtaining non-beaded and morphological accepted nanofibers. Altered concentrations of CA as cross-linker progressively enhanced significantly the mechanical/thermal stability and wettability-proof of NFs scaffolds, compared to un-cross-linked (PVA–Dex) scaffolds. Meanwhile, swelling (%), protein adsorption and released ampicillin of NFs decreased dramatically with the increase in the CA concentration, and conversely enhanced with increasing dextran concentrations. Interestingly, resultant PVA–Dex NFs with high concentrations of dextran promoted the proliferation of HFB-4 cells in a high concentration-dependent manner and high antimicrobial activity behavior, compared to NFs containing high concentrations of CA cross-linker after 24 and 48 h of cell exposure. Notably, all fabricated NFs have remarked ability to accelerate the rate of in vitro wound gap closure (%) after treatment for 24 and 48 h, compared to control sample. However, reducing CA concentration in NFs showed the highest percentages of wound healing for scratched HFB-4 cells with clear observed healing process.

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

Differentiation between Bacillus amyloliquefaciens and Bacillus subtilis isolated from a South African sugarcane processing factory using ARDRA and rpoB gene sequencing.

A total of 104 exopolysaccharide (gum)-producing bacteria were isolated from the juice screen and juice tank in a sugarcane processing factory at times of low- and high dextran concentrations in the produced sugar. Dextran is an indicator of cane deterioration and sucrose loss after harvesting of the cane. The isolates were identified as Bacillus amyloliquefaciens (96 isolates) and Bacillus subtilis (eight isolates) based on restriction enzyme banding patterns of amplified 16S rRNA genes and rpoB gene sequence analysis. Exopolysaccharide production in sugarcane is normally associated with dextran produced by Leuconostoc mesenteroides. B. amyloliquefaciens, and to a lesser extent B. subtilis, could, however, also be responsible for exopolysaccharide (slime or gum) production in cane processing factories.

Effects of Macromolecular Crowding on Alcohol Dehydrogenase and Citrate Synthase Kinetics

Most of our understanding of enzyme kinetics is based on experiments conducted in dilute solution. However, these conditions do not accurately represent the crowded cellular environment, which contains high concentrations (300–400g/L) of various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces the volume of solvent available for other molecules in the solution and has previously been shown to impact the behavior of enzymes. To study these potential consequences, the Michaelis‐Menten kinetics of yeast alcohol dehydrogenase (YADH) and citrate synthase (CS) were monitored under crowded conditions. Spectroscopic assays were performed in the presence and absence of high concentrations of dextran or glucose, the small‐molecule counterpart. This study also explored the effects of mixed crowding by combining equal ratios of different sizes of dextran. Overall, the effects of crowding depend on the size and concentration of the crowding agent. Furthermore, the results demonstrated that, in addition to merely excluded volume effects, chemical interactions from crowders must also be considered.Support or Funding InformationCamille and Henry Drefyus Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent

Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which they are studied. To better understand the ramifications of this crowding, the Michaelis-Menten kinetics of yeast alcohol dehydrogenase (YADH) were monitored spectrophotometrically in the presence of high concentrations of dextran. Crowding decreased the maximal rate of the reaction by 40% for assays with ethanol, the primary substrate of YADH. This observation was attributed to slowed release of the reduced β-nicotinamide adenine dinucleotide product, which is rate-limiting. In contrast, when larger alcohols were used as the YADH substrate, the rate-limiting step becomes hydride transfer and crowding instead increased the maximal rate of the reaction by 20-40%. This work reveals the importance of considering enzyme mechanism when evaluating the ways in which crowding can alter kinetics.

Macromolecular Crowding Effects on Two Homologs of Ribosomal Protein S16: Protein-Dependent Structural Changes and Local Interactions

Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Förster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16Meso, allowing measurements of three intraprotein distances. All S16Meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two S16Thermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

This paper investigates the thermodynamic and kinetic response of WT ⁎ ubiquitin (F45W) and three mutants to high concentrations of glucose, sucrose and dextran under physiological temperature and pH. WT ⁎ ubiquitin was stabilised by the same amount when comparing each cosolute on a weight to volume ratio, with cosolute effects largely independent of denaturant concentration. The energy difference between the mutants and WT ⁎ ubiquitin also remained the same in high concentrations of cosolute. An apparent decrease in transition-state surface burial in the presence of the cosolutes was attributed to increased compaction of the denatured state, and not to the Hammond effect. Together, these results suggest higher thermodynamic stabilities and folding rates for proteins in vivo compared to in vitro, in addition to more compact denatured states. Because the effects of mutation are the same in dilute solution and crowded conditions used to mimic the cellular environment, there is validity in using measurements of mutant stabilities made in dilute solutions to inform on how the mutations may affect stability in vivo.

Micro-rheological investigation of dextran solutions using diffusing wave spectroscopy

Diffusing-wave-spectroscopy-based micro-rheology was used to measure the rheological behaviour of dextran solutions of concentration up to 40%. Three dextrans of different molecular weights, namely 2×10 5, 5×10 5 and 2×10 6 Daltons, were studied. It was found that these solutions were mainly viscous, with the loss modulus G″ higher than the elastic modulus G′. However, at high frequencies and for high dextran concentrations, a crossover between G′ and G″ was observed. A plot of the specific viscosity η sp as a function of the dextran concentration showed two critical concentrations c* and c**. For c< c*, η sp ∼ c 1.4, for c*< c< c**, η sp ∼ c 2, and for c> c**, η sp ∼ c 3.8. These results were in very good agreement with those found in the literature.

Applications of time-resolved resonance energy transfer measurements in studies of the molecular crowding effect.

The native structures of many globular proteins are only weakly stabilized and form in solution ensembles of multiple conformers. The energy differences between the conformers are assumed to be small. This is the case of flexible multidomain proteins where domain motions were observed. High concentrations of inert macrosolute, which create a crowded or confined environment, can cause shifts of the distribution of the conformers of such proteins towards the more compact structures. This effect may also promote compact structures in partially folded proteins. Time-resolved dynamic non-radiative excitation energy transfer (tr-RET) is suitable for detection of either subtle or major changes in distributions of intramolecular distances in protein molecules in solutions. Two experiments were performed which demonstrated the applicability of tr-RET for detection of the effect of macrosolutes on the conformational ensembles of flexible states of protein molecules. The distribution of distances between residues 203 and 169 in the CORE domain of E. coli adenylate kinase (AK) in the denatured state was determined in the presence of high concentrations of dextran 40. A significant shift of the mean of the distribution was observed without reduction of its width. This was interpreted as a shift to compact structure without change of the degree of disorder of the chain. In a second experiment the distribution of the distance between residues 55 and 169 in AK, which spans the cleft between the CORE and the AMPbind domains, was monitored. No clear effect of high concentrations of dextran 40 was found. These experiments show the strength of the application of tr-RET in investigation of changes in the sub-states of flexible conformations of globular protein. Networks of pairs of labeled sites can be prepared and tr-RET experiments can be performed in order to search for the segments of the protein molecules, which respond to the presence of inert macromolecules in their environment.

Osmotic de-swelling of ionic microgel particles

In this work, we study experimentally the effect of an external osmotic pressure πext on the swelling of ionic mesoscopic gels in the weak screening limit, where the Debye screening length is larger than the mesh size of the gel. Variations in the osmotic pressure were induced by adding dextran to the solution. The results show that ionic microgels do not respond to πext below a given value of the normal stress; above this value the system de-swells with increasing osmotic pressure. The start of de-swelling is set by the gel charge density. The Flory thermodynamic theory for ionic gels captures the essential characteristics of the de-swelling behavior; in particular, it predicts with fairly good accuracy the value of πext at which de-swelling begins. Finally, due to the colloidal character of the gels, we observe that the system flocculates by a depletion interaction mechanism at high dextran concentrations.

Effect of Dextran on Protein Stability and Conformation Attributed to Macromolecular Crowding

Thermally induced transition curves of hen egg-white lysozyme were measured in the presence of several concentrations of dextran at pH 2.0 by near-UV and far-UV CD. The transition curves were fitted to a two-state model by a non-linear, least-squares method to obtain the transition temperature ( T m), enthalpy change (Δ H u( T m)), and free energy change (Δ G u( T)) of the unfolding transition. An increase in T m and almost constant Δ H u( T m) values were observed in the presence of added dextran at concentrations exceeding ca 100 g l −1. In addition, dextran-induced conformational changes of fully unfolded protein were investigated by CD spectroscopy. Addition of high concentrations of dextran to solutions of acid-unfolded cytochrome c at pH 2.0 results in a shift of the CD spectrum from that characteristic of the fully unfolded polypeptide to that characteristic of the more compact, salt-induced molten globule state, a result suggesting that the molten globule-like state is stabilized relative to the fully unfolded form in crowded environments. Both observations are in qualitative accord with predictions of a previously proposed model for the effect of intermolecular excluded volume (macromolecular crowding) on protein stability and conformation.

Macromolecular Crowding Accelerates Amyloid Formation by Human Apolipoprotein C-II

Human apolipoprotein C-II (apoC-II) slowly forms amyloid fibers in lipid-free solutions at physiological pH and salt concentrations (Hatters, D. M., MacPhee, C. E., Lawrence, L. J., Sawyer, W. H., and Howlett, G. J. (2000) Biochemistry 39, 8276--8283). Measurements of the time dependence of solution turbidity, thioflavin T reactivity, and the amount of sedimentable aggregate reveal that the rate and extent of amyloid formation are significantly increased by the addition of an inert polymer, dextran T10, at concentrations exceeding 20 g/liter. High dextran concentrations do not alter the secondary structure of the protein, fiber morphology, or the thioflavin T and Congo Red binding capacity of apoC-II amyloid. Analytical ultracentrifugation studies show that monomeric apoC-II does not associate significantly with dextran. The observed dependence of the overall rate of amyloid formation on dextran concentration may be accounted for quantitatively by a simple model for nonspecific volume exclusion. The model predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibers by any amyloidogenic protein.

Polymer Structure and Solubility of Deoxyhemoglobin S in the Presence of High Concentrations of Volume-excluding 70-kDa Dextran: EFFECTS OF NON-S HEMOGLOBINS AND INHIBITORS

Earlier observations indicated that volume exclusion by admixed non-hemoglobin macromolecules lowered the polymer solubility ("Csat") of deoxyhemoglobin (Hb) S, presumably by increasing its activity. In view of the potential usefulness of these observations for in vitro studies of sickling-related polymerization, we examined the ultrastructure, solubility behavior, and phase distributions of deoxygenated mixtures of Hb S with 70-kDa dextran, a relatively inert, low ionic strength space-filling macromolecule. Increasing admixture of dextran progressively lowered the Csat of deoxyHb S. With 12 g/dl dextran, a 5-fold decrease in apparent Csat ("dextran-Csat") was obtained together with acceptable sensitivity and proportionality with the standard Csat when assessing the effects of non-S Hb admixtures (A, C, and F) or polymerization inhibitors (alkylureas or phenylalanine). The volume fraction of dextran excluding Hb was 70-75% of total deoxyHb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-to-crystal transitions indistinguishable from those formed without dextran. Thus when Hb quantities are limited, as with genetically engineered recombinant Hbs or transgenic sickle mice, the dextran-Csat provides convenient and reliable screening of effects of Hb S modifications on polymerization under near-physiological conditions, avoiding problems of high ionic strength.

The influence of locust bean gum and dextran on the gelation of κ-carrageenan

The interaction of κ-carrageenan with locust bean gum and dextran has been studied by rheology, differential scanning calorimetry (DSC), and electron spin resonance spectroscopy (ESR). Rheological measurements show that the carrageenan gel characteristics are greatly enhanced in the presence of locust bean gum but not in the presence of dextran. Carrageenan/locust bean gum mixtures show two peaks in the dsc cooling curves. The higher temperature peak corresponds to the temperature of gelation and its intensity increases at the expense of the lower temperature peak as the proportion of locust bean gum in the mixture increases. Furthermore, the DSC heating curves show enhanced broadening when locust bean gum is present, indicating increased aggregation. These results are taken as evidence of carrageenan/locust bean gum association. The gelation process has also been followed by ESR using spin-labeled carrageenan. On cooling carrageenan solutions, an immobile component appears in the ESR spectra signifying a loss of segmental mobility consistent with chain stiffening due to the coil → helix conformational transition and helix aggregation. For carrageenan/locust bean gum mixtures, carrageenan ordering occurs at temperatures corresponding to the higher temperature DSC setting peak and the temperature of gelation. Similar studies using spin-labeled locust bean gum show that its mobility remains virtually unaffected during the gelation process. It is evident, therefore, that carrageenan and locust bean gum interact only weakly. It is proposed that at low carrageenan concentrations the gel network consists of carrageenan helices cross-linked by locust bean gum chains. At high carrageenan concentrations the network is enhanced by the additional self-aggregation of the “excess” carrageenan molecules. For carrageenan/dextran mixtures, only one peak is observed in the dsc cooling curves. The onset of gelation shifts to higher temperatures only at very high (20%) dextran concentrations and this is attributed to volume exclusion effects. Furthermore, there is no enhanced broadening of the peaks in the DSC heating curves as for the carrageenan/locust bean gum systems. It is therefore concluded that carrageenan/dextran association does not occur. The difference in behavior between locust bean gum and dextran is attributed to the greater flexibility of the dextran chains. © 1996 John Wiley & Sons, Inc.

Analysis of Fluorophore Diffusion by Continuous Distributions of Diffusion Coefficients: Application to Photobleaching Measurements of Multicomponent and Anomalous Diffusion

Fluorescence recovery after photobleaching (FRAP) is widely used to measure fluorophore diffusion in artificial solutions and cellular compartments. Two new strategies to analyze FRAP data were investigated theoretically and applied to complex systems with anomalous diffusion or multiple diffusing species: 1) continuous distributions of diffusion coefficients, α( D), and 2) time-dependent diffusion coefficients, D( t). A regression procedure utilizing the maximum entropy method was developed to resolve α( D) from fluorescence recovery curves, F( t). The recovery of multi-component α( D) from simulated F( t) with random noise was demonstrated and limitations of the method were defined. Single narrow Gaussian α( D) were recovered for FRAP measurements of thin films of fluorescein and size-fractionated FITC-dextrans and Ficolls, and multi-component α( D) were recovered for defined fluorophore mixtures. Single Gaussian α( D) were also recovered for solute diffusion in viscous media containing high dextran concentrations. To identify anomalous diffusion from FRAP data, a theory was developed to compute F( t) and α( D) for anomalous diffusion models defined by arbitrary nonlinear mean-squared displacement 〈 x 2〉 versus time relations. Several characteristic α( D) profiles for anomalous diffusion were found, including broad α( D) for subdiffusion, and α( D) with negative amplitudes for superdiffusion. A method to deduce apparent D( t) from F( t) was also developed and shown to provide useful complementary information to α( D). α( D) and D( t) were determined from photobleaching measurements of systems with apparent anomalous subdiffusion (nonuniform solution layer) and superdiffusion (moving fluid layer). The results establish a practical strategy to characterize complex diffusive phenomena from photobleaching recovery measurements.

The influence of thermodynamic activity on the solute rejection in multicomponent systems

In this study it has been shown that major changes in solute rejection can occur when the solute is part of a multicomponent system instead of a binary system. The effect of changes in thermhermodynamic activity of the solute on its rejection has been studied. As a model system the combined ultrafiltration of poly(ethylene glycol) (PEG) and dextran has been chosen. The thermodynamic activities of PEG and dextran have been modelled with the UNIQUAC model. Rejection measurements showed a decrease in the observed PEG rejection when dextran was added to the solutions under conditions where the dextran was mainly retained by the membrane. At high dextran concentrations and low fluxes the PEG rejection even became strongly negative. The PEG concentration in the permeate was at maximum a factor 2 higher than in the permeate (on a dextran-free basis). For a more open membrane the rejection of PEG was found to increase in the presence of dextran. The elevated transport of PEG through the membrane in the presence of dextran was ascribed to the additional driving force due to the increased thermodynamic activity of PEG at the retentate side of the membrane. The results in this paper show that thermodynamic interactions can have a strong impact on the observed rejection and are certainly worth being considered in multicomponent systems.

Dextran as protectant against damage caused by sparging for hybridoma cells in a bubble column

The effect of the addition of dextran as a protective polymer against sparging was examined with hybridoma suspension cells in a bubble column under standardized conditions. The protective effect of high concentrations of high molecular weight dextran showed a correlation with the bulk viscosity of the medium. A distinct protective effect occurs at viscosities greater than 20 × 10 −3 Pa s −1. In contrast, low molecular weight dextrans that cause a minor increase in viscosity, also provide no protection against sparging. There is no strict correlation between surface tension and the protective effect of dextran against sparging. Oxygen transfer is strongly reduced by high concentrations of high molecular weight dextran. Therefore, addition of dextran as protective polymer against sparging for large-scale production processes with animal cells in stirred reactors does not seem feasible.

Topo-optical investigations of human erythrocyte glycocalyx conformational changes induced by dextran

Cell surface properties are involved in the aggregation process of red blood cells. Using the topo-optical toluidine blue reaction, conformational changes of the glycocalyx (main component glycophorin A) were found when red blood cells were incubated and fixed in the presence of dextran. Relative differences in optical path as a measure of red blood cell membrane anisotropy decreased in relation to dextran concentration during fixation. These conformational changes could not be detected by electrophoretic measurements. When incubating, fixing and staining red blood cells in the presence of dextran, anisotropy decreased only at low dextran concentrations and increased at rising dextran concentrations. This biphasic course of differences in optical path seems to be due to different effects of dextran superimposing upon each other: a disturbing influence on the spatial order of sialic acid carrying oligosaccharide side chains due to H-bond interaction, and an increase in the size of dye aggregates and suppression of the thermal motion of macromolecules at higher dextran concentrations.

The incompatibility of concentrated aqueous solutions of dextran and amylose and its effect on amylose gelation

At 75°C concentrated aqueous solutions of dextran and amylose exhibit immiscibility. The presence of dextran has a major effect on the microstructure and mechanical behaviour of amylose gels formed on cooling. At low dextran concentrations the modulus of the gel increases with increasing dextran concentration, whereas at higher dextran concentrations, polymer incompatibility and segregation of dextran-rich droplets reduces the firmness of the gel.