Reversible pore block of connexin channels by cyclodextrins

Cyclodextrins (CDs), a series of hollow cyclic glucosaccharides, can reversibly block molecular permeation through channels formed by connexin-32 and/or connexin-26 reconstituted into liposomes. The character of the block changes as a function of the size of the CD relative to the connexin pore diameter, suggesting that the block occurs via entry of the CD into the pore lumen and occlusion of the permeability pathway. The block occurs only when the CD is applied to the side of the pore that is normally cytoplasmic and not from the side that is normally extracellular. The block is potentiated when organic analytes are sequestered in the hydrophobic interior of the CDs. CDs may be useful as molecular tools with which to explore the structure of the connexin pore and to alter molecular movement through connexin channels.     

Spectrofluorometric analytical applications of cyclodextrins

Cyclodextrins (CDs) are a family of cyclic oligosaccharides composed of α‐(1,4)‐linked glucopyranose subunits. The most important feature of CDs is their ability to form inclusion complexes (host–guest complexes) with a very wide range of solid, liquid and gaseous compounds by a molecular complexation. During the last decade, a considerable number of research papers has been focused on the use of CDs to enhance fluorescence intensity of different analytes and to develop CD‐induced spectrofluorimetric method. In this review, the various spectrofluorimetric methods based on host–inclusion complex are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

The effect of cyclodextrins on the ethanol tolerance of microorganisms suggests potential application

Cyclodextrins (CDs) are used in food, pharmaceutical, and chemical industries, as well as agriculture and environmental engineering. Cyclodextrin glucanotransferase (CGTase) is an important industrial extracellular enzyme which is used to produce CDs and oligosaccharides. We previously developed a novel yeast-surface CGTase expression system which was used for the production of CDs from starch. In the present study, we showed that the presence of CDs may increase the ethanol tolerance of microorganisms. The cell numbers of Saccharomyces cerevisiae and Escherichia coli in the presence of β-cyclodextrin and ethanol were 1,000-fold and 10-fold higher than that without CDs. The yeast strain with the immobilized CGTase produced 13 g CDs/l and 1.8 g ethanol/l when it was incubated in yeast medium supplemented with 4% starch. The effect of CDs on microorganisms suggests a potential application for the co-production of CDs and ethanol.     

Evidence for the 2:1 molecular recognition and inclusion behaviour between beta- and gamma-cyclodextrins and cinchonine

Cinchonine (Cin) is the primary drug of choice in the treatment of malaria, but its poor solubility has restricted its use via the oral route. Cyclodextrins (CDs) form inclusion complexes with cinchonine to form soluble complexes. This interaction was investigated by solubility studies, electrospray ionization mass spectrometry (ESI-MS), and molecular modeling. ESI-MS evaluated successfully the nature of the solution-phase inclusion complexes. The experimental results showed that not only 1:1, but also stable 2:1 inclusion complexes can be formed between CDs and Cin. Multi-component complexes of beta-CD-Cin-beta-CD (1:1:1), gamma-CD-Cin-gamma-CD (1:1:1), and beta-CD-Cin-gamma-CD (1:1:1) were found in equimolar beta- and gamma-CD mixtures with Cin. The formation of 2:1 and multi-component 1:1:1 non-covalent CD-Cin complexes indicates that beta- and gamma-CD are able to form sandwich-type inclusion complexes with Cin in high concentrations. The phase-solubility diagram showed non-linear type A(p) profile, indicating that more than one cyclodextrin molecule is involved in the complexation of one guest molecule. Molecular modeling calculations have been carried out to rationalize the experimental findings and predict the lowest energy molecular structure of inclusion complex.     

Biotechnological applications of cyclodextrins

Cyclodextrins (CDs) are a family of cyclic oligosaccharides that are composed of alpha-1,4-linked glucopyranose subunits. Cyclodextrins are produced from starch by enzymatic degradation. These macrocyclic carbohydrates with apolar internal cavities can form complexes with and solubilize many normally water-insoluble compounds. This review describes recent applications of CDs in pharmaceuticals with a major emphasis on drug delivery systems. The utility of these water-soluble cyclic glucans in a variety of foods, flavors cosmetics, packaging and textiles is elaborated. The role of these compounds in biocatalysis is also discussed. Cyclodextrins are used in separation science because they have been shown to discriminate between positional isomers, functional groups, homologues and enantiomers. This property makes them a useful agent for a wide variety of separations.     

Enantioselective toxicity of metolachlor to Scenedesmus obliquus in the presence of cyclodextrins

Cyclodextrins (CDs) possess a variety of chiral centers and are capable of recognizing enantiomeric molecules through the formation of inclusion complexes. Two types of CDs, α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD), were selected to evaluate the effects of the enantioselective ecotoxicity of racemic metolachlor (Rac-metolachlor) and its S-enantiomer (S-metolachlor) on the freshwater algae Scenedesmus obliquus (S. obliquus) by acute toxicity test. The results showed that the aquatic toxicity of S-metolachlor was higher than Rac-metolachlor and that CDs enhanced the toxicity of metolachlor enantioselectively by increasing the aquatic toxicity of Rac-metolachlor rather than that of S-metolachlor to S. obliquus. The equilibrium constant for Rac-metolachlor-CD complexes was higher than that of S-metolachlor-CDs, which was responsible for the greater aquatic toxicity shift effect of Rac-metolachlor. Thermodynamic studies of CD complexes showed that inclusion for all of the complexes was primarily a spontaneous, enthalpy-driven process. These results will help to understand the preliminary mechanism of shifting aquatic toxicity of metolachlor by CDs and the CDs mediated environmental processes of metolachlor, to correctly apply CDs to chiral pesticides formulation and environmental remediation of chiral contaminants.     

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol-rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen-glucose deprivation (OGD), N-methyl-D-aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only beta-, methylated beta- and sulfated beta-CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD-95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X-100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated beta-CD to protect against NMDA toxicity, and reversed NR2B, PSD-95 and nNOS localization to Triton X-100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor-mediated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), but did suppress OGD-induced increases in [Ca(2+)](i). beta-CD, but not Mbeta-CD, also caused a slight block of NMDA-induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent-resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol-rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca(2+) levels.     

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.     

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.     

A new technique for promoting cyclic utilization of cyclodextrins in biotransformation

Cyclodextrins (CDs) can improve the productivity of steroid biotransformation by enhancing substrate solubility. CDs can be recycled by grafting them with appropriate carriers. Loofah fiber is an excellent grafting material for CDs, and can be applied to the biotransformation and recycling of β-cyclodextrin (β-CD). In this work, a technique for recycling β-CD in cortisone acetate (CA) biotransformation by Arthrobacter simplex CPCC 140451 was studied. Loofah fiber-grafted β-CD (LF-β-CD) was prepared using epichlorohydrin, which is a cross-linking agent. The grafting yield of β-CD was 74.8 mg g^?1 dried fibers. LF-β-CD could increase the solubility of CA and enhance biotransformation. The initial conversion rate of CA was 1.5-fold higher than that of the blank group. LF-β-CD was also used in biocatalytic reactions for eight cycles, and it maintained the conversion ratio of CA at approximately 90%. Given the above positive results, LF-β-CD can be utilized in biotechnological recycling applications. This method can also be applied to CD derivatives and hydrophobic compounds.     

Cyclodextrin induces calcium-dependent lysosomal exocytosis

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders.     

Chlorin p6 as a fluorescent probe for the investigation of surfactant-cyclodextrin interactions.

Cyclodextrins (CD) are often proposed as potential vehicles in targeted drug delivery. However, if the membrane structure is disrupted by CD, then it cannot be considered to be a good drug delivery vehicle. When an extrinsic fluorescence probe is used to monitor such interactions, there are no less than three possible equilibria that can operate simultaneously: surfactant-cyclodextrin, surfactant-fluorophore and cyclodextrin-fluorophore. The fluorescence intensity/lifetime might be affected by all these and so, the results depend strongly on the fluorophore used as well as the nature of the surfactant. This aspect highlights the importance of the suitability of the fluorescence probe to be used to study complicated systems and interaction. In the present work, chlorin p6, prepared from chlorophyll from spinach leaves, has been used as the fluorescence probe to investigate the interaction between alpha-CD and beta-CD with the neutral surfactants Triton X 100 (TX 100) and cetyl trimethyl ammonium bromide (CTAB). The fluorophore is found to be a sensitive one for the study of the interaction of alpha, beta and gamma-CD with the surfactants TX 100 and CTAB. It is found that contrary to earlier reports, a complex between alpha-CD and TX 100 is formed, even though the binding constant is not very high. This observation can be obtained with chlorin p6, which does not bind to the CDs, but not with a fluorophore, which binds to the CD as well and thus complicates the situation as the binding with CD is stronger than that between TX 100 and alpha-CD as compared to that between TNS and CD.     

Lysozyme refolding with cyclodextrins: structure-activity relationship

Cyclodextrins (CDs), in the presence or absence of detergents, have been reported to suppress aggregate formation during the refolding of a number of proteins. A structure-activity relationship study between CD chemistry and refolding of lysozyme was performed and compared to carbonic anhydrase, in order to better understand the mechanism of CD-assisted protein refolding and to identify CDs that could function as good protein folding agents. Among the natural CDs, which have only hydroxyl groups, alpha-CD, with a smaller cavity size was more effective than the oligosaccharide with a larger cavity, gamma-CD. Replacement of the hydroxyls with other functional groups did not improve, but could seriously interfere, with the lysozyme refolding ability of alpha-CD. In case of gamma-CD, substitution of its hydroxyls with other groups either enhanced or diminished its refolding capability towards lysozyme. In general, neutral CDs were better refolding agents than the charged sugars. The presence of anionic substituents like carboxyl and phosphate groups actually promoted aggregate formation and completely abolished the sugar's refolding ability. This effect was more pronounced with lysozyme than with carbonic anhydrase. CDs with cationic functional groups did not show any significant effects on lysozyme refolding. The presence of both anionic and cationic substituents on the same CD molecule was found to partially restore its renaturation ability. Electrophoresis data indicate that CDs, which promoted lysozyme refolding, arrested aggregation at the stage of smaller soluble aggregates. Interestingly, the structure-activity relationship observed with lysozyme was quite similar to that reported for a non-disulfide protein, carbonic anhydrase. These results suggest that the effects of CDs on protein refolding are attributed to their ability to suppress aggregation of proteins. CDs may show properties similar to chaotropic agents, which may help explain their anti-aggregation and protein refolding ability. Besides alpha-CD, a number of other neutral CDs were found to be effective protein folding aids.     

Apo alpha-lactalbumin and lysozyme are colocalized in their subsequently formed spherical supramolecular assembly

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.     

Two independent ways of preparing hypercharged hydrolyzable polyaminorotaxane

The aim of this work is to synthesize new PEO-based polyrotaxanes from modified cyclodextrins. Two strategies are discussed and compared. In the first, a pseudopolyrotaxane was formed between alpha,omega- PEO dimethacrylate and alpha-cyclodextrin. A coupling reaction between 1-pyrenebutyric acid N-hydroxysuccinimide ester was carried out to block the cyclic molecules onto the PEO. Cyclodextrins of the supramolecular assemblies were then oxidized using sodium periodate and reacted with spermine to form a potentially highly charged polyrotaxane. In the second strategy, cyclodextrins were first modified, and used to form the polyrotaxane through the pseudopolyrotaxane synthesis followed by the blocking reaction. Acidic titration allowed quantifying the number of amine functions borne by the supramolecular assemblies through two variables: the number of rings per polymer chain and the number of spermine groups per cyclic molecule. The supramolecules obtained by both strategies are discussed.     

Refolding of chemically denatured alpha-amylase in dilution additive mode

Cyclodextrins (CDs) possess hydrophobic surfaces, which probably shield the hydrophobic surfaces of denatured proteins and prevent the direct interactions between the surfaces which are believed to be responsible for protein aggregation during refolding process. This probability was evaluated by studying the refolding process of denatured alpha-amylase in the presence and absence of alpha-CD, as a dilution additive agent. Our data indicate that in the presence of 100 mM alpha-CD in the refolding buffer, the extent of aggregation reduces by almost 90%. Spectrofluorometric analysis of the refolding intermediate(s) also indicates that the tertiary structure of the refolded alpha-amylase, in the presence of alpha-CD, is very similar to the tertiary structure of the native protein. However, this similarity was distorted upon addition of exogenous hydrophobic (aliphatic or aromatic) amino acids to the refolding buffer, meaning that the hydrophobic interactions between alpha-CD and the denatured protein play significant role in preventing aggregate formation. In addition, by weakening the extent of these hydrophobic interactions by adding polarity-reducing agent (e.g. ethylene glycol) to the refolding buffer, more aggregates were formed. In contrast, strengthening these interactions by enhancing the ionic strength of the refolding buffer made these hydrophobic interactions very strong. Therefore, alpha-CD could not depart from the protein/alpha-CD complex, as it usually does during the process of refolding. As a result, more aggregates were formed in the presence of alpha-CD compared to the corresponding control samples.     

Multiscale structures of lipids in foods as parameters affecting fatty acid bioavailability and lipid metabolism

On a nutritional standpoint, lipids are now being studied beyond their energy content and fatty acid (FA) profiles. Dietary FA are building blocks of a huge diversity of more complex molecules such as triacylglycerols (TAG) and phospholipids (PL), themselves organised in supramolecular structures presenting different thermal behaviours. They are generally embedded in complex food matrixes. Recent reports have revealed that molecular and supramolecular structures of lipids and their liquid or solid state at the body temperature influence both the digestibility and metabolism of dietary FA. The aim of the present review is to highlight recent knowledge on the impact on FA digestion, absorption and metabolism of: (i) the intramolecular structure of TAG; (ii) the nature of the lipid molecules carrying FA; (iii) the supramolecular organization and physical state of lipids in native and formulated food products and (iv) the food matrix. Further work should be accomplished now to obtain a more reliable body of evidence and integrate these data in future dietary recommendations. Additionally, innovative lipid formulations in which the health beneficial effects of either native or recomposed structures of lipids will be taken into account can be foreseen.     

Rhodopsin activation follows precoupling with transducin: inferences from computational analysis

The electrostatic and shape complementarities between the crystal structures of dark rhodopsin and heterotrimeric transducin (Gt) have been evaluated by exhaustively sampling the roto-translational space of one protein with respect to the other. Structural complementarity, reliability, and consistency with in vitro evidence all converge in the same rhodopsin-Gt complex, showing that the functionally important R135 of the E/DRY motif is almost accessible to the C-terminus of Gt(alpha) already in the dark state. The main inference from this study is that activation of rhodopsin and Gt may be concurrent processes, consisting of conformational changes in a supramolecular complex formed prior to the light-induced activation of the photoreceptor.     

Actin-Based Gelation of Cytoplasmic Extracts from Brain Tissue

Incubation of rat brain cytoplasmic extracts under the conditions described in this paper results in the formation of three-dimensional gels. Ultrastructurally, these gels correspond to complex supramolecular structures formed by single microfilaments and by microfilament bundles. Analysis of protein composition indicates that cytoplasmic gels are composed of actin and several associated proteins. Among the latter class of components, we have identified polypeptides with molecular weights of 55,000 (55K), 140,000 (140K), and a set of two or three polypeptides with molecular weights in the range of 235,000-245,000 (235-245K). The 55K and 140K components do not seem to correspond to any previously identified actin-associated proteins, while the 235-245K polypeptides may correspond to the protein known as fodrin.     

The role of xanthophylls in the supramolecular organization of the photosynthetic complex LHCII in lipid membranes studied by high-resolution imaging and nanospectroscopy

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular β-structures. Meanwhile, the molecules of violaxanthin act as “molecular spacers” preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.