Comparing the Effects of Encapsulated and Non-Encapsulated Propolis Extracts on Model Lipid Membranes and Lactic Bacteria, with Emphasis on the Synergistic Effects of Its Various Compounds.

Propolis is a resinous compound made by bees with well-known biological activity. However, comparisons between encapsulated and non-encapsulated propolis are lacking. Therefore, the antibacterial activity, effect on the phase transition of lipids, and inhibition of UV-induced lipid oxidation of the two forms of propolis were compared. The results showed that non-encapsulated propolis produces quicker effects, thus being better suited when more immediate effects are required (e.g., antibacterial activity). In order to gain an in-depth introspective on these effects, we further studied the synergistic effect of propolis compounds on the integrity of lipid membranes. The knowledge of component synergism is important for the understanding of effective propolis pathways and for the perspective of modes of action of synergism between different polyphenols in various extracts. Thus, five representative molecules, all previously isolated from propolis (chrysin, quercetin, trans-ferulic acid, caffeic acid, (-)-epigallocatechin-3-gallate) were mixed, and their synergistic effects on lipid bilayers were investigated, mainly using DSC. The results showed that some compounds (quercetin, chrysin) exhibit synergism, whereas others (caffeic acid, t-ferulic acid) do not show any such effects. The results also showed that the synergistic effects of mixtures composed from several different compounds are extremely complex to study, and that their prediction requires further modeling approaches.

Thermal and Rheological Properties of Gluten-Free, Starch-Based Model Systems Modified by Hydrocolloids.

Obtaining good-quality gluten-free products represents a technological challenge; thus, it is important to understand how and why the addition of hydrocolloids influences the properties of starch-based products. To obtain insight into the physicochemical changes imparted by hydrocolloids on gluten-free dough, we prepared several suspensions with different corn starch/potato starch/hydroxpropyl methyl cellulose/xanthan gum/water ratios. Properties of the prepared samples were determined by differential scanning calorimetry and rheometry. Samples with different corn/potato starch ratios exhibited different thermal properties. Xanthan gum and HPMC (hydroxypropyl methyl cellulose) exhibited a strong influence on the rheological properties of the mixtures since they increased the viscosity and elasticity. HPMC and xanthan gum increased the temperature of starch gelatinization, as well as they increased the viscoelasticity of the starch model system. Although the two hydrocolloids affected the properties of starch mixtures in the same direction, the magnitude of their effects was different. Our results indicate that water availability, which plays a crucial role in the starch gelatinization process, could be modified by adding hydrocolloids such as, hydroxypropyl methyl cellulose and xanthan gum. By adding comparatively small amounts of the studied hydrocolloids to starch, one can achieve similar thermo-mechanical effects by the addition of gluten. Understanding these effects of hydrocolloids could contribute to the development of better quality gluten-free bread with optimized ingredient content.

Interactions of (-)-epigallocatechin-3-gallate with model lipid membranes.

(-)-Epigallocatechin-3-gallate (EGCG) is a flavonoid known for its good antioxidant potential and health benefits. It is one of the most intriguing flavonoids, especially because of its specific interactions with model lipid membranes. It was noticed that EGCG might form EGCG rich domains/rafts at certain compositions of lipid membranes. In this article, we investigate whether EGCG forms EGCG rich domains when incorporated in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes. Our results show that EGCG decreases lipid ordering parameter in ordered membranes and increases it in the case of disordered ones. Also, incorporation of EGCG does not affect the zeta-potential and shape of the liposomes, but it can induce aggregation of liposomes. Our study also demonstrates that liposomes with incorporated EGCG are highly protected against UV-light induced oxidation.

Thermally Induced Transitions of d(G4T4G3) Quadruplexes Can Be Described as Kinetically Driven Processes.

DNA sequences that are rich in guanines and can form four-stranded structures are called G-quadruplexes. Due to the growing evidence that they may play an important role in several key biological processes, the G-quadruplexes have captured the interest of several researchers. G-quadruplexes may form in the presence of different metal cations as polymorphic structures formed in kinetically governed processes. Here we investigate a complex polymorphism of d(G4T4G3) quadruplexes at different K+ concentrations. We show that population size of different d(G4T4G3) quadruplex conformations can be manipulated by cooling rate and/or K+ concentration. We use a kinetic model to describe data obtained from DSC, CD and UV spectroscopy and PAGE experiments. Our model is able to describe the observed thermally induced conformational transitions of d(G4T4G3) quadruplexes at different K+ concentrations.

Contribution of headgroup and chain length of glycerophospholipids to thermal stability and permeability of liposomes loaded with calcein.

Biological membranes are complex systems that are composed of lipids, proteins and carbohydrates. They are difficult to study, so it is established practice to use lipid vesicles that consist of closed 'shells' of phospholipid bilayers as model systems to study various functional and structural aspects of lipid organisation. To define the effects of the structural properties of lipid vesicles on their phase behaviour, we investigated their headgroup and chain length, and the chemical bonds by which their acyl chains are attached to the glycerol moiety of glycerophospholipid species, in terms of phase transition temperature, enthalpy change and calcein permeability. We used differential scanning calorimetry to measure the temperature and enthalpy changes of phase transition, and fluorescence to follow calcein release through the bilayer structure. Our data show that longer acyl chains increase the stability of the lipid bilayers, whereas higher salt concentrations decrease the thermal stability and widen the phase transitions of these lipid bilayers. We discuss the possible reasons for the observed phase transition behaviour.

Proof of concept web application for understanding the energetic basis of oligonucleotide unfolding.

Measuring and quantifying thermodynamic parameters that determine both the stability of and interactions between biological macromolecules are an essential and necessary complement to structural studies. Although basic thermodynamic parameters for an observed process can be readily obtained, the data interpretation is often slow and analysis quality can be extremely variable. We have started to develop a web application that will help users to perform thermodynamic characterizations of oligonucleotide unfolding. The application can perform global fitting of calorimetric and spectroscopic data, and uses a three-state equilibrium model to obtain thermodynamic parameters for each transition step - namely, the Gibbs energy, the enthalpy, and the heat capacity. In addition, the application can define the number of K+ ions and the number of water molecules being released or taken up during unfolding. To test our application, we used UV spectroscopy, circular dichroism, and differential scanning calorimetry to monitor folding and unfolding of a model 22-nucleotide-long sequence of a human 3'-telomeric overhang, known as Tel22. The obtained data were uploaded to the web application and the global fit revealed that unfolding of Tel22 involves at least one intermediate state, and that K+ ions are released during the unfolding, whereas water molecules are taken up.

Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome.

Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.

The size of the internal loop in DNA hairpins influences their targeting with partially complementary strands.

Targeting of noncanonical DNA structures, such as hairpin loops, may have significant diagnostic and therapeutic potential. Oligonucleotides can be used for binding to mRNA, forming a DNA/RNA hybrid duplex that inhibits translation. This kind of modulation of gene expression is called the antisense approach. In order to determine the best strategy to target a common structural motif in mRNA, we have designed a set of stem-loop DNA molecules with sequence: d(GCGCTnGTAAT5GTTACTnGCGC), where n = 1, 3, or 5, "T5" is an end loop of five thymines. We used a combination of calorimetric and spectroscopy techniques to determine the thermodynamics for the reaction of a set of hairpins containing internal loops with their respective partially complementary strands. Our aim was to determine if internal- and end-loops are promising regions for targeting with their corresponding complementary strands. Indeed, all targeting reactions were accompanied by negative changes in free energy, indicating that reactions proceed spontaneously. Further investigation showed that these negative free energy terms result from a net balance of unfavorable entropy and favorable enthalpy contributions. In particular, unfolding of hairpins and duplexes is accompanied by positive changes in heat capacity, which may be a result of exposure of hydrophobic groups to the solvent. This study provides a new method for the targeting of mRNA in order to control gene expression.

A new pathway of DNA G-quadruplex formation.

A new folding intermediate of Oxytricha nova telomeric Oxy-1.5 G-quadruplex was characterized in aqueous solution using NMR spectroscopy, native gel electrophoresis, thermal differential spectra (TDS), CD spectroscopy, and differential scanning calorimetry (DSC). NMR experiments have revealed that this intermediate (i-Oxy-1.5) exists in two symmetric bimolecular forms in which all guanine bases are involved in GG N1-carbonyl symmetric base pairs. Kinetic analysis of K(+) -induced structural transitions shows that folding of Oxy-1.5 G-quadruplex from i-Oxy-1.5 is much faster and proceeds through less intermediates than folding from single strands. Therefore, a new folding pathway of Oxy-1.5 G-quadruplex is proposed. This study provides evidence that G-rich DNA sequences can self-assemble into specific pre-organized DNA structures that are predisposed to fold into G-quadruplex when interacting with cations such as potassium ions.

Energetic basis of human telomeric DNA folding into G-quadruplex structures.

Recent theoretical studies performed on the folding/unfolding mechanism of the model telomeric human DNA, 5'-AGGGTTAGGGTTAGGGTTAGGG-3' (Tel22), have indicated that in the presence of K(+) ions Tel22 folds into two hybrid G-quadruplex structures characterized by one double and two reversal TTA loops arranged in a different way. They predicted a new unfolding pathway from the initial mixture of hybrid G-quadruplexes via the corresponding intermediate triplex structures into the final, fully unfolded state. Significantly, no experimental evidence supporting the suggested pathway has been reported. In the current work, we performed a comprehensive global thermodynamic analysis of calorimetric (DSC, ITC) and spectroscopic (CD) data obtained on monitoring the folding/unfolding of Tel22 induced by changes of temperature and K(+) concentration. We show that unfolding of Tel22 may be described as a monomolecular equilibrium three-state process that involves thermodynamically distinguishable folded (F), intermediate (I), and unfolded (U) state. Considering that calorimetric methods cannot distinguish between energetically similar G-quadruplex or triplex conformations predicted by the theoretical model one can conclude that our results represent the first experimental support of the suggested unfolding/folding mechanism of Tel22. This conclusion is confirmed by the fact that the estimated number of K(+) ions released upon each unfolding step in our thermodynamic model agrees well with the corresponding values predicted by the theoretical model and that the observed changes in enthalpy, entropy, and heat capacity accompanying the F → I and I → U transitions can be reasonably explained only if the intermediate state I is considered to be a triplex structural conformation.

Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function.

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

Kinetically Governed Formation of d(G4T2G4) Assemblies.

The most frequently appearing form of DNA is a double helical structure in which two single strands are held together by Watson-Crick base pairs. In addition, guanine rich DNA sequences are known to adopt several unusual structures with G-quartet as a basic repeating motif. Recently large self-assembling nanostructures, called G-wires have become of great interest because of their potential use in molecular electronics. To better understand the forces driving structural transitions of G-wires formed from d(G4T2G4) oligonucleotide in Na+ solutions, we employed a number of techniques such as UV and CD spectroscopy, differential scanning calorimetry (DSC), gel electrophoresis, small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). All the experimental techniques showed that thermally induced folding transitions depend on the cooling rate. In addition, DSC shows that thermally induced unfolding transition depends on heating rate, thus leading to the conclusion that structural transitions of d(G4T2G4) are kinetically governed processes.

Interaction of minor groove ligands with G-quadruplexes: thermodynamic contributions of the number of quartets, T-U substitutions, and conformation.

In the presence of specific metal ions, DNA oligonucleotides containing guanine repeat sequences can adopt G-quadruplex structures. In this work, we used a combination of spectroscopic and calorimetric techniques to investigate the conformation and unfolding thermodynamics of the K(+)-form of five G-quadruplexes with sequences: d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), G2, d(G(3)T(2)G(3)TGTG(3)T(2)G(3)), G3, their analogs where T is replaced with U, G2-U and G3-U, and r(G(2)U(2)G(2)UGUG(2)U(2)G(2)), rG2. These G-quadruplexes show CD spectra characteristic of the "chair" conformation (G2 and G2-U), or "basket" conformation (rG2); or a mixture of these two conformers (G3 and G3-U). Thermodynamic profiles show that the favorable folding of each G-quadruplex results from the typical compensation of a favorable enthalpy and unfavorable entropy contributions. G-quadruplex stability increase in the following order (in ΔG°(20)): rG2 (-1.3 kcal/mol) < G2 < G2-U

Kinetically governed polymorphism of d(G4T4G3) quadruplexes in K+ solutions.

It has been generally recognized that understanding the molecular basis of some important cellular processes is hampered by the lack of knowledge of forces that drive spontaneous formation/disruption of G-quadruplex structures in guanine-rich DNA sequences. According to numerous biophysical and structural studies G-quadruplexes may occur in the presence of K(+) and Na(+) ions as polymorphic structures formed in kinetically governed processes. The reported kinetic models suggested to describe this polymorphism should be considered inappropriate since, as a rule, they include bimolecular single-step associations characterized by negative activation energies. In contrast, our approach in studying polymorphic behavior of G-quadruplexes is based on model mechanisms that involve only elementary folding/unfolding transitions and structural conversion steps that are characterized by positive activation energies. Here, we are investigating a complex polymorphism of d(G(4)T(4)G(3)) quadruplexes in K(+) solutions. On the basis of DSC, circular dichroism and UV spectroscopy and polyacrylamide gel electrophoresis experiments we propose a kinetic model that successfully describes the observed thermally induced conformational transitions of d(G(4)T(4)G(3)) quadruplexes in terms of single-step reactions that involve besides single strands also one tetramolecular and three bimolecular quadruplex structures.

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate.

Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC-MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 microg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 microg/mL and LOQ was 0.5 microg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC-MS/MS(n), Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity.

Diverse polymorphism of G-quadruplexes as a kinetic phenomenon.

Knowledge of forces that drive conformational transitions of G-quadruplexes is crucial for understanding the molecular basis of several key cellular processes. It can only be acquired by combining structural, thermodynamic and kinetic information. Existing biophysical and structural evidences on polymorphism of intermolecular G-quadruplexes have shown that the formation of a number of these structures is a kinetically controlled process. Reported kinetic models that have been used to describe the association of single strands into quadruplex structures seem to be inappropriate since the corresponding model-predicted activation energies turn out to be negative. By contrast, we propose here a novel kinetic model that successfully describes experimentally monitored folding/unfolding transitions of G-quadruplexes and gives positive activation energies for all elementary steps, including those describing association of two single strands into bimolecular quadruplex structures. It is based on a combined thermodynamic and kinetic investigation of polymorphic behavior of bimolecular G-quadruplexes formed from d(G4T4G4) and d(G4T4G3) strands in the presence of Na(+) ions, monitored by spectroscopic (UV, CD) and calorimetric (DSC) techniques. According to our experiment and model analysis the topology of the measured G-quadruplexes is clearly flexible with the conformational forms that respond to the rate of temperature change at which global unfolding/folding transitions occur.

Erythropoietin unfolding: thermodynamics and its correlation with structural features.

Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state.