Effects of beta-cyclodextrin addition and temperature on the modulation of hydrophobic interactions in aqueous solutions of an associative alginate

Novel information about the effects of beta-cyclodextrin (beta-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HM-alginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of beta-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high beta-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of beta-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between beta-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of beta-CD suggested that the polymer chains are locally stretched at all of the considered levels of beta-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that beta-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate.     

Thermoreversible gelation of aqueous mixtures of pectin and chitosan. Rheology

The synergistic interaction between pectin and chitosan in aqueous acid solution and in the gel phase has been studied by oscillatory shear measurements. Mixtures of pectin and chitosan form thermoreversible gels over a broad composition range by lowering the temperature. The value of the gelation temperature depends on the composition of the mixture, with low values for mixtures with low pectin contents. For incipient gels, a power law can describe the frequency dependence of the complex viscosity, with power law exponents close to -1. The gel evolution of pectin-chitosan mixtures upon a temperature quench below the gel point has been studied. Evidence is provided for a relation between gelation and phase separation in the process of temperature-induced gelation of pectin-chitosan mixtures. A simple model is proposed to rationalize the gelation process in these systems.     

Rheological characterization of photochemical changes of ethyl(hydroxyethyl)cellulose dissolved in water in the presence of an ionic surfactant and a photosensitizer

The effects of addition of the photosensitizer riboflavin (RF) to semidilute solutions of the systems ethyl(hydroxyethyl)cellulose (EHEC)/water, EHEC/sodium dodecyl sulfate (SDS), and EHEC/cetyltrimethylammonium bromide (CTAB) on the turbidity and the linear viscoelasticity are studied. The turbidity behavior and the cloud point (CP) are influenced by the addition of RF to the EHEC/SDS system, whereas no discernible change is observed for the other systems. The rheological features of all systems are affected by the presence of RF at lower temperatures, whereas at temperatures close to the CP, only a slight effect is detected. Both the EHEC/SDS and EHEC/CTAB systems evolve thermoreversible gels at the same temperature (37.5 degrees C), but in the presence of RF, the EHEC/CTAB system does not form a gel, whereas the gel temperature for the EHEC/SDS system is depressed (32.5 degrees C). Light irradiation of RF in the EHEC/SDS/RF system causes fragmentation of the network and a higher temperature is required to re-form the incipient gel network. The photochemical degradation of EHEC gives rise to a decrease in the dynamic moduli and the complex viscosity for all of the three systems. The effect is strengthened at higher temperatures and it is most pronounced for the EHEC/SDS system.     

Characterization of the chemical degradation of hyaluronic acid during chemical gelation in the presence of different cross-linker agents

Dynamic light scattering (DLS) and rheological experiments have been performed on semidilute aqueous hyaluronic acid (HA) solutions during the chemical cross-linking process with a water-soluble carbodiimide (WSC) to produce a hydrogel. The formation and destruction of the gel are characterized. The results suggest that the gel is cross-linked via ester linkages and at later stage in the process, the omnipresent hydrolysis of interpolymer ester linkages and glycosidic bonds prevails, leading to disruption of the gel. The process of forming and breaking the gel is affected by the cross-linker concentration and pH. The cross-linking of HA with WSC in the presence of L-lysine methyl ester produced a gel with a longer time of gelation and the degradation of the gel was prolonged because of the more stable amide bond formation as the cross-link. By using the Ugi multicomponent condensation reaction, interpolymer cross-linking occurs via the formation of amide linkages and a stable gel evolves, which is only slightly degraded over an extended time window. DLS measurements on HA solutions with WSC show the emergence of a long-time power-law tail in the correlation functions at conditions both before and beyond the viscosity maximum. At a late stage in the gel-breakage regime, the power-law profile of the decay disappears and the long-time tail of the correlation function can be portrayed by a stretched exponential. The findings indicate that the power-law feature is associated with the confinement of chain dynamics and anomalous diffusion in the system. At later times, the connectivity is lost due to fragmentation of the network, and the long-time stretched exponential decay in the correlation function reflects the relaxation of clusters of various sizes.     

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.     

Influence of chain length and branching on poly(ADP-ribose)–protein interactions

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR–protein interactions. Quantitative K_d values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53—and potentially also APLF—can form complex multivalent PAR–protein structures. In conclusion, our study gives detailed and quantitative insight into PAR–protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.     

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Acetate as the major by-product in industrial-scale bioprocesses with Escherichia coli is found to decrease process efficiency as well as to be toxic to cells, which has several effects like a significant induction of cellular stress responses. However, the underlying phenomena are poorly explored. Therefore, we studied time-resolved population heterogeneity of the E. coli growth reporter strain MG1655/pGS20PrrnBGFPAAV expressing destabilized green fluorescent protein during batch growth on acetate and glucose as sole carbon sources. Additionally, we applied five fluorescent stains targeting different cellular properties (viability as well as metabolic and respiratory activity). Quantitative analysis of flow cytometry data verified that bacterial populations in the bioreactor are more heterogeneous in growth as well as stronger metabolically challenged during growth on acetate as sole carbon source, compared to growth on glucose or acetate after diauxic shift. Interestingly, with acetate as sole carbon source, significant subpopulations were found with some cells that seem to be more robust than the rest of the population. In conclusion, following batch cultures population heterogeneity was evident in all measured parameters. Our approach enabled a deeper study of heterogeneity during growth on the favored substrate glucose as well as on the toxic by-product acetate. Using a combination of activity fluorescent dyes proved to be an accurate and fast alternative as well as a supplement to the use of a reporter strain. However, the choice of combination of stains should be well considered depending on which population traits to aim for.     

Immunomodulatory activity of commonly used drugs on Fc-receptor-mediated human natural killer cell activation

Natural killer (NK) cells mediate defense against neoplastic as well as infected cells. Yet, how their effector functions are affected by the large variety of pharmacological compounds commonly in use has not been investigated systematically. Here, we screened 1,200 in-use or previously approved drugs for their biological effect on freshly isolated human peripheral blood-derived NK cells. Mimicking antibody-dependent cellular cytotoxicity (ADCC), known to be important in antibody-based immunotherapies against, e.g., human malignancies, the cells were stimulated by Fc-receptor (CD16) engagement. Cellular responses were assessed by flow cytometry. Fifty-six compounds that significantly inhibited and twelve that enhanced one or more of the readouts of adhesion, exocytosis, and chemokine production were identified and confirmed as hits. Among the confirmed inhibitors, 80 % could be assigned to one of seven major pharmacological classes. These classes were β2-adrenergic agonists, prostaglandins, phosphodiesterase-4 inhibitors, Ca²⁺-channel blockers, histamine H1-receptor antagonists, serotonin/dopamine receptor antagonists, and topoisomerase inhibitors that displayed distinct inhibitory patterns on NK cell responses. Among observed enhancers, interestingly, two ergosterol synthesis inhibitors were identified that specifically promoted exocytosis. In summary, these results provide a comprehensive knowledge base of the effect known drugs have on NK cells. More specifically, they provide an overview of drugs that may modulate NK cell-mediated ADCC in the context of clinical immunotherapies.