Effect of platinum(II) chemotherapeutic agents on properties of DNA liquid crystals

We have investigated the X-ray and optical properties (CD spectra and polarization microscopy) of liquid-crystalline phases and dispersions formed on pretreatment of low molecular weight DNA with the platinum(II) coordination complexes, cis-diammine-dichloroplatinum(II) (DDP), 2,2'-bipyridinedichloroplatinum(II) (1) and 2,2'-bipyridineethylenediammineplatinum(II) (2). It is demonstrated that the platination of DNA leads to the ordering of neighbouring molecules of DNA in liquid-crystalline phases being diminished. The intense bands observed in the CD spectra of liquid-crystalline dispersions prepared from DNA pretreated with 1 or 2 can be used to determine the orientation of the latter compounds with respect to the helical axis of the DNA and to detect distortions in the secondary structure of DNA. The possible causes of the appearance of the intense bands in the CD spectra of liquid-crystalline phases and alterations in the manner of packing of the molecules of DNA within them are discussed.     

Extreme stability of helices formed by water-soluble poly-N-substituted glycines (polypeptoids) with alpha-chiral side chains.

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.     

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.     

Solvent selectivity in chiral chromatography using a β-cyclodextrin-bonded phase

A β-cyclodextrin-bonded phase has been used to investigate the separation of the enantiomers of atenolol, oxprenolol, celiprolol, tertatolol, terbutaline, fluoxetine, norfluoxetine, and zopiclone, focusing on the importance of solvent selectivity. With cyclodextrin (CD)-bonded phases, chiral discrimination occurs because the two enantiomers of a racemate form inclusion complexes of different strengths within the CD cavity. The organic modifier molecules tend to compete with solutes for a definite number of adsorption sites on the stationary phase. Moreover, the ternary complex formation may play an important role in chiral recognition. In this study, it was of interest to estimate the influence of mobile phase modifiers with respect to solvent type (i.e., ACN, MeOH, EtOH, THF, i-PrOH, PrOH and t-BuOH), size and shape, and concentration. Solvent selectivity has been investigated by using different organic modifiers in mobile phases with the same polarity, and relationships were established between the logarithm of solvent partition coefficient (log P_s) and the three most important chromatographic parameters: retention time (t), resolution (R), and enantioselectivity (α). Thus, it seems that the hydrophobicity of the organic modifier becomes one of the dominant factors affecting the inclusion process phenomena. Further, the apparent partition coefficients of the compounds under study have been determined and a comparison has been attempted regarding the degree of their enantiomeric resolution. © 1995 Wiley-Liss, Inc.     

Regulation of the ryanodine receptor calcium release channel: a molecular complex system

The thermal behaviour and structural changes associated with the phase transformation of 1,2-dipalmitoyl-sn-glycerol (DPG) were studied by means of simultaneous X-ray diffraction and differential scanning calorimetry. Metastable DPG solid phases are crystallized from the melted sample by thermal quenching. The metastable phase (alpha-phase) formed initially is converted into a stable phase (beta' phase) at approximately 50 degrees C on heating. It was found that the behaviour of the alpha- to beta'-phase transformation depends on the thermal history. DPG solid samples incubated at approximately 3 degrees C for more than 10 h after cooling transformed directly into the beta'-phase with heat release. On the other hand, in the solid samples without incubation, the alpha-phase once melted and then the crystallization of the beta'-phase occurred successively from the melted state.     

Thermal analysis of marginal conditions to facilitate cryopreservation by vitrification using a semi-empirical approach

This study aims at the thermal analysis of marginal conditions leading to cryopreservation by vitrification, which appears to be the only alternative for indefinite preservation of large-size tissues and organs. The term “marginal conditions” here refers to cooling rates in close range with the so-called critical cooling rate, above which crystallization is avoided. The analysis of thermal effects associated with partial crystallization during vitrification is associated with the coupled phenomena of heat transfer and kinetics of crystallization. This study takes a practical, semi-empirical approach, where heat transfer is analyzed based on its underlying theoretical principles, while the thermal effects associated with partial crystallization are taken into account by means of empirical correlations. This study presents a computation framework to solve the coupled problem, while presenting a proof-of-concept for DP6 as a representative cryoprotective agent. The thermal effects associated with crystallization at various relevant cooling rates are measured in this study by means of differential scanning calorimetry. Results of this study demonstrate that, due to the thermal effects associated with partial crystallization, the cooling rate at the center of a large organ may lag behind the cooling rate in its surroundings under some scenarios, but may also exceed the surroundings cooling rate in other scenarios, leading to counter-intuitive effects associated with partial crystallization.     

Phospholipid metabolism of 3T3 mouse fibroblasts after serum stimulation and through the Gl and S cell cycle phases : incorporation and disappearance of P

Phospholipid metabolism of 3T3 mouse fibroblasts has been studied after serum stimulation of arrested cells.The study of [³²Pi]incorporation shows : a) in the case of PE and PC an early peak of incorporation in the Gl phase of the cell cycle, 6 hours after serum addition ; b) in the case of PI an intense initial increasing of the incorporation which continues up to a S phase peak.The study of the disappearance of [³²P] Phosphate from the different phospholipids points out : a) at the beginning of serum stimulation, an intense breakdown of PI, that continues through the Gl and S phases. Except at the onset, the breakdown of PI, is at any time exactly compensated for by synthesis : the two phenomena are closely linked ; b) a synthesis of PE, from PC probably, at the Gl phase, 4 hours after the serum addition and the beginning of the chase experiment.     

Review of biomaterial thermal property measurements in the cryogenic regime and their use for prediction of equilibrium and non-equilibrium freezing applications in cryobiology

It is well accepted in cryobiology that the temperature history and cooling rates experienced in biomaterials during freezing procedures correlate strongly with biological outcome. Therefore, heat transfer measurement and prediction in the cryogenic regime is central to the field. Although direct measurement of temperature history (i.e. heat transfer) can be performed, accuracy is usually achieved only for local measurements within a given system and cannot be readily generalized to another system without the aid of predictive models. The accuracy of these models rely upon thermal properties which are known to be highly dependent on temperature, and in the case of significant cryoprotectant loading, also on crystallized fraction. In this work, we review the available thermal properties of biomaterials in the cryogenic regime. The review shows a lack of properties for many biomaterials in the subzero temperature domain, and especially for systems with cryoprotective agents. Unfortunately, use of values from the limited data available (usually only down to −40 °C) lead to an underestimation of thermal property change (i.e. conductivity rise and specific heat drop due to ice crystallization) with lower temperatures. Conversely, use of surrogate values based solely on ice thermal properties lead to an overestimation of thermal property change for most biomaterials. Additionally, recent work extending the range of available thermal properties to −150 °C has shown that the thermal conductivity will drop in both PBS and tissue (liver) due to amorphous/glassy phases (versus crystalline) of biomaterials with the addition of cryoprotective additives such as glycerol. Thus, we investigated the implications of using approximated or constant property values versus measured temperature-dependent values for predicting temperature history during freezing in PBS (phosphate-buffered saline) and porcine liver with and without cryoprotectants (glycerol). Using measured property values (thermal conductivity, specific heat, and latent heat of phase change) of porcine liver, a standard was created which showed that values based on surrogate ice properties under-predicted cooling times, while constant properties (i.e. based on limited data reported near the freezing point) over-predicted cooling times. Additionally, a new iterative numerical method that accommodates non-equilibrium cooling effects as a function of time and position (i.e. crystallization versus amorphous phase) was used to predict temperature history during freezing in glycerol loaded systems. Results indicate that in addition to the increase in cooling times due to the lowering of thermal diffusivity with more glycerol, non-equilibrium effects such as the prevention of maximal crystallization (i.e. amorphous phases) will further increase required cooling times. It was also found that the amplified effect of non-equilibrium cooling and crystallization with system size prevents the thermal history to be described with non-dimensional lengths, such as was possible under equilibrium cooling. These results affirm the need to use accurate thermal properties that incorporate temperature dependence and crystallized fraction. Further studies are needed to extract thermal properties of other important biomaterials in the subzero temperature domain and to develop accurate numerical methods which take into account non-equilibrium cooling events encountered in cryobiology when partial or total vitrification occurs.     

Destabilization of mayonnaise induced by lipid crystallization upon freezing

The thermal and rheological history of mayonnaise during freezing and its dispersion stability after the freeze-thaw process were investigated. Mayonnaise was cooled to freeze and stored at ?20 to ?40 °C while monitoring the temperature; penetration tests were conducted on the mayonnaise, which was sampled at selected times during isothermal storage at ?20 °C. Significant increases in the temperature and stress values due to water-phase crystallization and subsequent oil-phase crystallization were observed. The water phase crystallized during the cooling step in all the tested mayonnaise samples. The oil phases of the prepared mayonnaise (with rapeseed oil) and commercial mayonnaise crystallized during isothermal storage after 6 and 4 h, respectively, at ?20 °C. The dispersion stability was evaluated from the separation ratio, which was defined as the weight ratio of separated oil after centrifuging to the total amount of oil in the commercial mayonnaise. The separation ratio rapidly increased after 4 h of freezing. This result suggests that crystallization of the oil phase is strongly related to the dispersion stability of mayonnaise.     

CsI‐Antisolvent Adduct Formation in All‐Inorganic Metal Halide Perovskites

The excellent optoelectronic properties demonstrated by hybrid organic/inorganic metal halide perovskites are all predicated on precisely controlling the exact nucleation and crystallization dynamics that occur during film formation. In general, high‐performance thin films are obtained by a method commonly called solvent engineering (or antisolvent quench) processing. The solvent engineering method removes excess solvent, but importantly leaves behind solvent that forms chemical adducts with the lead‐halide precursor salts. These adduct‐based precursor phases control nucleation and the growth of the polycrystalline domains. There has not yet been a comprehensive study comparing the various antisolvents used in different perovskite compositions containing cesium. In addition, there have been no reports of solvent engineering for high efficiency in all‐inorganic perovskites such as CsPbI₃. In this work, inorganic perovskite composition CsPbI₃ is specifically targeted and unique adducts formed between CsI and precursor solvents and antisolvents are found that have not been observed for other A‐site cation salts. These CsI adducts control nucleation more so than the PbI₂–dimethyl sulfoxide (DMSO) adduct and demonstrate how the A‐site plays a significant role in crystallization. The use of methyl acetate (MeOAc) in this solvent engineering approach dictates crystallization through the formation of a CsI–MeOAc adduct and results in solar cells with a power conversion efficiency of 14.4%.     

Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.     

Conformational changes of alamethicin induced by solvent and temperature. A 13C-NMR and circular-dichroism study.

13C nuclear magnetic resonance (NMR) and circular dichroism (CD) have been used for studies on the conformation of alamethicin. The 13C NMR spectrum is assigned with the aid of signals of synthetic partial sequences and selective proton decoupling. The solvent and temperature-dependence of the 13C NMR spectra, T1 measurements and the use of lanthanide-shift reagents allow the differentiation between the amino acids belonging to a rigid alpha-helical portion of the alamethicin sequence and those belonging to a more flexible part. The 13C NMR results are in agreement with results obtained from extended solvent and temperature-dependent CD studies which indicate a highly stabilized nonpolar and intrachenar alpha-helical part. The concentration-dependence of the CD spectrum of alamethicin in a nematic phase revealed aggregation phenomena which might simulate those observed in natural and synthetic membranes. After dissolving alamethicin in aqueous alcohol there is a time-dependence of the ellipticity of the Cotton effects showing a sort of memory effect on the mode of dissolution. Four different conformations can be characterized by CD spectra depending on the solvent and concentration. A model illustrating the dynamic conformations and aggregation phenomena within a membrane is proposed.     

Isotropic-nematic phase equilibrium and phase separation of kappa-carrageenan in aqueous salt solution: experimental and theoretical approaches

The behavior of chiral-nematic and isotropic phases of helical kappa-carrageenan in aqueous solution of sodium iodide was compared with that of the anisotropic biphasic phase that contains both these phases. On the basis of birefringence, rheology, chemical analysis, average molecular weight, and polydispersity index measurements, we derived a number of characteristic differences as well as similarities between these phases, over a range of polysaccharide concentrations obtained by the dilution of each phase. For example, we assessed the critical concentration of an isotropic-anisotropic transition (C(i)), the temperature of the anisotropic-isotropic phase shift during thermal heating-cooling cycles, and the viscosity changes due to the phase shift and due to the diminishing of the helical conformation. We also demonstrated how the different phases and their dilutions behave under the effect of shear and frequency of oscillation and how the viscoelastic properties vary in each phase and discussed the isotropic and anisotropic liquid crystal controlling behavior mechanisms. From a theoretical point of view, we propose to combine the wormlike chain model for semiflexible polyelectrolytes interacting via both hard-core and electrostatic repulsion to assess the concentration of isotropic-nematic transition, to assess the coexistence concentration range, and to determine the effects of charge by applying the effective diameter and a twisting effect.     

Phase and glass transition behaviour of concentrated barley starch-glycerol-water mixtures, a model for thermoplastic starch

The effects of glycerol and water content on the thermal transitions of plasticized barley starch were examined using differential scanning calorimetry. The glycerol contents studied were 14, 20, 29 and 39% and the water content, obtained by conditioning in different relative humidities, varied in the range 1–28%. On the basis of the observed calorimetric glass transition temperatures and corresponding heat capacity increments it was inferred that a single phase system occurred at low water and glycerol contents, while in other cases phase separation occurred and the system was composed of starch-rich and starch-poor phases. Dynamic mechanical thermal analysis on a phase-separated sample showed mechanical loss peaks corresponding to the glass transitions of both phases. Amylopectin crystallization did not occur within 1 week of storage in mixtures having less than 20% water, indicating that glycerol interacted with starch, inhibiting crystallization of amylopectin.     

Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase

It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H(+)/K(+)-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H(+)/K(+)-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H(+)/K(+)-ATPase.     

Solvent-induced beta-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central -Aib-Gly- segment capable of adopting beta-turn conformations compatible with both hairpin (beta(II') or beta(I')) and helical (beta(I)) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined beta-hairpin conformation in solvents capable of hydrogen bonding like (CD(3))(2)SO and CD(3)OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl(3) and CD(3)CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl(3) and (CD(3))(2)SO by 500 MHz (1)H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl(3). In (CD(3))(2)SO, peptide VII adopts a beta-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition.     

Helix–coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(γ-benzyl-L-glutamate-co-L-alanine) and poly(γ-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host–guest technique has been applied to the determination of the helix–coil stability constants of two naturally occurring amino acids, L -alanine and L -leucine, in a nonaqueous solvent system. Random copolymers containing L -alanine and L -leucine, respectively, as guest residues and γ-benzyl-L -glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix–coil transition behavior in a dichloroacetic acid (DCA)–1,2-dichloroethane (DCE) mixture. Two types of helix–coil transitions were carried out on the copolymers: solvent-induced transitions in DCA–DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA–DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, σ and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L -alanine residue stabilizes the α-helical conformation more than the L -leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.     

Solvent effects on the conformation and far UV CD spectra of gramicidin

Solvent effects on the far-uv CD spectra of the polypeptide gramicidin have been studied systematically in a series of alcohols of increasing chain length, ranging from methanol to dodecanol. The effects observed are of two types: primary, involving a change in the equilibrium mixture of conformers present, and secondary, involving a shift in the spectral peak positions as a function of solvent polarizability. To quantitate the primary effect, the ratio of the individual conformers present was estimated by deconvolution of the spectra into their component species. For short chain length alcohols, both parallel and antiparallel double helices are found in considerable abundance. As the solvent chain length is increased and its polarity is decreased, the left-handed antiparallel double helical species is favored. For all alcohols with chain lengths of four or more carbon atoms, the ratio of the conformers present remains relatively constant. To quantitatively examine the secondary effect, the magnitudes of the spectral shifts on the dominant conformer (species 3) have been correlated with the dielectric constants and refractive indices of the solvents, thereby indicating what underlying physical properties are responsible for these shifts. This work thus demonstrates that for gramicidin, a flexible polypeptide, the solvent effects on the CD spectra can be resolved into two types: changes due to the mixture of conformers present and shifts in the spectral characteristics. Both effects need to be considered when interpreting CD spectra in terms of secondary structure for this and other polypeptides in nonaqueous solutions.