Curcumin Modulates 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) Liposomes: Chitosan Oligosaccharide Lactate Influences Membrane Fluidity But Does Not Alter Phase Transition Temperature of DBPC Liposomes.

1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) is one of the important phospholipids found in cell membrane but not studied well. Importance of curcumin as a dietary supplement and for its medicinal properites is getting widely recoginsed. The present study for the first time explores the effect of curcumin on properties of DBPC liposomes by monitoring the fluorescence properties of curcumin. The phase transition temperature (Tm) of DBPC is assessed which confirms increase in curcumin concentration causes a slight drop in the Tm value. Chitosan is being applied for various drug delivery uses. The study establishes new insight on effect of chitosan oligosaccharide lactate on DBPC liposomes. It is found that in the absence of chitosan oligosaccharide lactate, curcumin partitions more strongly in the liquids crystalline phase than in the solid gel phase, however, the opposite result is obtained with the presence of chitosan oligosaccharide lactate which penetrates into the DBPC liposomes membranes at higher temperature, blocking thus the passage of curcumin into the lipid bilayer. However, addition of chitosan oligosaccharide lactate had no effect on the Tm. Fluorescence quenching study of curcumin establishes that the location of curcumin to be in the hydrophobic cavity of DBPC membrane.

Dual mechanism of ionic liquid-induced protein unfolding.

Ionic liquids (ILs) are gaining attention as protein stabilizers and refolding additives. However, varying degrees of success with this approach motivates the need to better understand fundamental IL-protein interactions. A combination of experiment and simulation is used to investigate the thermal unfolding of lysozyme in the presence of two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO4] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et2PO4]). Both ILs reduce lysozyme melting temperature Tm, but more gradually than strong denaturants. [EMIM][Et2PO4] lowers lysozyme Tm more readily than [EMIM][EtSO4], as well as requiring less energy to unfold the protein, as determined by the calorimetric enthalpy ΔH. Intrinsic fluorescence measurements indicate that both ILs bind to tryptophan residues in a dynamic mode, and furthermore, molecular dynamics simulations show a high density of [EMIM]+ near lysozyme's Trp62 residue. For both ILs approximately half of the [EMIM]+ cations near Trp62 show perfect alignment of their respective rings. The [EMIM]+ cations, having a "local" effect in binding to tryptophan, likely perturb a critically important Arg-Trp-Arg bridge through favorable π-π and cation-π interactions. Simulations show that the anions, [EtSO4]- and [Et2PO4]-, interact in a "global" manner with lysozyme, due to this protein's strong net positive charge. The anions also determine the local distribution of ions surrounding the protein. [Et2PO4]- is found to have a closer first coordination shell around the protein and stronger Coulomb interactions with lysozyme than [EtSO4]-, which could explain why the former anion is more destabilizing. Patching of ILs to the protein surface is also observed, suggesting there is no universal IL solvent for proteins, and highlighting the complexity of the IL-protein environment.

Synthesis of Glycosidic (ß-1''→6, 3' and 4') Site Isomers of Neomycin B and their Effect on RNA and DNA Triplex Stability.

Glycosidic (ß-1''→6, 3' and 4') site isomers of neomycin B (i.e., neobiosamine (ß-1''→6, 3' and 4') neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3',4'-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4'-, 5,3',4'- and 5,6,3'-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.

Application of a Salt Coformer in a Co-Amorphous Drug System Dramatically Enhances the Glass Transition Temperature: A Case Study of the Ternary System Carbamazepine, Citric Acid, and l-Arginine.

The use of co-amorphous systems containing a combination of low molecular weight drugs and excipients is a relatively new technology in the pharmaceutical field to improve the solubility of poorly water-soluble drugs. However, some co-amorphous systems show a lower glass transition temperature ( Tg) than many of their polymeric solid dispersion counterparts. In this study, we aimed at designing a stable co-amorphous system with an elevated Tg. Carbamazepine (CBM) and citric acid (CA) were employed as the model drug and the coformer, respectively. co-amorphous CBM-CA at a 1:1 molar ratio was formed by ball milling, but a transition from the glassy to the supercooled melt state was observed under ambient conditions, due to the relatively low Tg of 38.8 °C of the co-amorphous system and moisture absorption. To improve the Tg of the coformer, salt formation of a combination of l-arginine (ARG) with CA was studied. First, ball milling of CA-ARG at molar ratios of 1:1, 1:2, and 1:3 forming co-amorphous systems was performed and led to a dramatic enhancement of the Tg, depending on the CA-ARG ratio. Salt formation between CA and ARG was observed by infrared spectroscopy. Next, ball milling of CBM-CA-ARG at molar ratios of 1:1:1, 1:1:2, and 1:1:3 resulted in co-amorphous blends, which had a single Tg at 77.8, 105.3, and 127.8 °C, respectively. These ternary co-amorphous samples remained in a solid amorphous form for 2 months at 40 °C. From these results, it can be concluded that blending of the salt coformer with a drug is a promising strategy to design stable co-amorphous formulations.

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions.

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6M concentrations compared to only 1M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.

Laurdan emission study of the cholesterol-like effect of long-chain alkylresorcinols on the structure of dipalmitoylphosphocholine and sphingomyelin membranes.

Long-chain alkylresorcinols (ARs) are commonly found in plant and bacteria cells, and they exhibit a wide variety of biological effects, including antifungal, antitumor, and antiphrastic activities. The cholesterol (Chol)-like effect of ARs with hydrocarbon side-chain lengths ranging from C15 to C25 on the structure of pure and Chol-doped dipalmitoylphosphocholine (DPPC) and sphingomyelin (SM) membranes was investigated by Laurdan fluorescence spectroscopy. The Laurdan emission generalized polarization parameter was analyzed as a function of the temperature and excitation wavelength in DPPC (or SM)/Chol, DPPC (or SM)/AR, and DPPC/Chol/AR systems. It was found that AR incorporation into both DPPC and SM bilayers induces an increase in the temperature of the main lipid phase transition, similar to the effect of Chol molecule incorporation. The phase separation, lipid-chain ordering, and membrane hydration are discussed for the AR-mixed membranes and compared with DPPC (or SM)/Chol membranes.

Probing the relationship between anti-Pneumocystis carinii activity and DNA binding of bisamidines by molecular dynamics simulations.

The anti-Pneumocystis carinii activity of 13 synthetic pentamidine analogs was analyzed. The experimental differences in melting points of DNA dodecamer 5'-(CGCGAATTCGCG)2-3' complexes (ΔTm), and in the biological activity measured using ATP bioluminescence assay (IC50) together with the theoretical free energy of DNA-ligand binding estimated by the proposed computational protocol, showed that the experimental activity of the tested pentamidines appeared to be due to the binding to the DNA minor groove with extended AT sequences. The effect of heteroatoms in the aliphatic linker, and the sulfonamide or methoxy substituents on the compound inducing changes in the interactions with the DNA minor groove was examined and was correlated with biological activity. In computational analysis, the explicit solvent approximation with the discrete water molecules was taken into account, and the role of water molecules in the DNA-ligand complexes was defined.

A dihydroindolizino indole derivative selectively stabilizes G-quadruplex DNA and down-regulates c-MYC expression in human cancer cells.

BACKGROUND: Telomeric and NHE III1, a c-MYC promoter region is abundant in guanine content and readily form G-quadruplex structures. Small molecules that stabilize G-quadruplex DNA were shown to reduce oncoprotein expression, initiate apoptosis and they may function as anticancer molecules. METHODS: Electrospray ionization mass spectrometry, spectroscopy, isothermal titration calorimetry, Taq DNA polymerase stop assay, real time PCR and luciferase reporter assay. Cell migration assay to find out the effect of derivatives on normal as well as cancer cell proliferation. RESULTS: Among three different dihydroindolizino indole derivatives, 4-cyanophenyl group attached derivative has shown maximum affinity, selective interaction and higher stability towards G-quadruplex DNA over dsDNA. Further, as a potential G-quadruplex DNA stabilizer, 4-cyanophenyl linked dihydroindolizino indole derivative was found to be more efficient in inhibiting in vitro DNA synthesis, c-MYC expression and cancer cell proliferation among human cancer cells. CONCLUSION: The present study reveals that dihydroindolizino indole derivative having 4-cyanophenyl group has potential to stabilize G-quadruplex DNA and exhibit anticancer activity. GENERAL SIGNIFICANCE: These studies are useful in the identification and synthesis of lead derivatives that will selectively stabilize G-quadruplex DNA and function as anticancer agents.

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1µM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner.

Quantifying the temperature dependence of glycine-betaine RNA duplex destabilization.

Glycine-betaine (GB) stabilizes folded protein structure because of its unfavorable thermodynamic interactions with amide oxygen and aliphatic carbon surface area exposed during protein unfolding. However, GB can attenuate nucleic acid secondary structure stability, although its mechanism of destabilization is not currently understood. Here we quantify GB interactions with the surface area exposed during thermal denaturation of nine RNA dodecamer duplexes with guanine-cytosine (GC) contents of 17-100%. Hyperchromicity values indicate increasing GB molality attenuates stacking. GB destabilizes higher-GC-content RNA duplexes to a greater extent than it does low-GC-content duplexes due to greater accumulation at the surface area exposed during unfolding. The accumulation is very sensitive to temperature and displays characteristic entropy-enthalpy compensation. Since the entropic contribution to the m-value (used to quantify GB interaction with the RNA solvent-accessible surface area exposed during denaturation) is more dependent on temperature than is the enthalpic contribution, higher-GC-content duplexes with their larger transition temperatures are destabilized to a greater extent than low-GC-content duplexes. The concentration of GB at the RNA surface area exposed during unfolding relative to bulk was quantified using the solute-partitioning model. Temperature correction predicts a GB concentration at 25 °C to be nearly independent of GC content, indicating that GB destabilizes all sequences equally at this temperature.

Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe.

Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 µM. Treatment with 0, 250, 500 or 1000 µM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 µM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.

Development, characterisation and biocompatibility testing of a cobalt-containing titanium phosphate-based glass for engineering of vascularized hard tissues.

There is a continuing need to develop scaffold materials that can promote vascularisation throughout the tissue engineered construct. This study investigated the effect of cobalt oxide (CoO) doped into titanium phosphate glasses on material properties, biocompatibility and vascular endothelial growth factor (VEGF) secretion by osteoblastic MG63 cells. Glasses composed of (P2O5)45(Na2O)20(TiO2)05(CaO)30-x(CoO)x(x=0, 5, 10, and 15 mol%) were fabricated and the effect of Co on physicochemical properties including density, glass transition temperature (Tg), degradation rate, ion release, and pH changes was assessed. The results showed that incorporation of CoO into the glass system produced an increase in density with little change in Tg. It was then confirmed that the pH did not change significantly when CoO was incorporated in the glass, and stayed constant at around 6.5-7.0 throughout the dissolution study period of 336 h. Ion release results followed a specific pattern with increasing amounts of CoO. In general, although incorporation of CoO into a titanium phosphate glass increased its density, other bulk and surface properties of the glass did not show any significant changes. Cell culture studies performed using MG63 cells over a 7-day period indicated that the glasses provide a stable surface for cell attachment and are biocompatible. Furthermore, VEGF secretion was significantly enhanced on all glasses compared with standard tissue culture plastic and Co doping enhanced this effect further. In conclusion, the developed Co-doped glasses are stable and biocompatible and thus offer enhanced potential for engineering vascularized tissue.

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes.

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (Tm) of the peptide-RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as L-2-amino-4-guanidinobutyric acid (Agb) or L-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the L-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide-RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.

DNA duplex stabilization in crowded polyanion solutions.

The melting temperature of duplex DNA is much higher in polyanions than in non-ionic polymers with similar ionic strength, suggesting an additional electrostatic contribution on top of the excluded volume effect.

Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase.

Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H2O2 into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25 °C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (Tm) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.

DNA binding, antioxidant activity, and DNA damage protection of chiral macrocyclic Mn(III) salen complexes.

We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral complexes showed ability to bind with DNA, where complex S-1 exhibits the highest DNA binding constant 1.20 × 10(6) M(-1). All the compounds were screened for superoxide and hydroxyl radical scavenging activities; among them, complex S-1 exhibited significant activity with IC(50) 1.36 and 2.37 µM, respectively. Further, comet assay was used to evaluate the DNA damage protection in white blood cells against the reactive oxygen species wherein complex S-1 was found effective in protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage.

The crucial role of divalent metal ions in the DNA-acting efficacy and inhibition of the transcription of dimeric chromomycin A3.

Chromomycin A3 (Chro) is capable of forming a stable dimeric complex via chelation with Ni(II), Fe(II) and Co(II). According to the circular dichroism study, the dimer conformations are significantly different among the Fe(II)-, Co(II)-, and Ni(II)-containing dimeric Chro complexes; however, the dimer conformations were preserved at high temperatures. Furthermore, we conducted a systematic study to determine the effects of these divalent metal ions on the DNA-acting efficacy of dimeric Chro, including its DNA-binding affinity, DNA stabilization capacity, DNA cleavage activity, and the inhibition of transcription both in vitro and within cells. Kinetic analyses using surface plasmon resonance (SPR) showed that Ni(II)(Chro)(2) exhibited the highest K(a) with a value of 1.26 × 10(7) M(-1), which is approximately 1.6- and 3.7-fold higher than the K(a) values obtained for Co(II)(Chro)(2) and Fe(II)(Chro)(2), respectively. The T(m) and ΔG values for the DNA duplex increased after the addition of drug complexes in the following order: Ni(II)(Chro)(2)>Co(II)(Chro)(2)>Fe(II)(Chro)(2). In the DNA integrity assays, the DNA cleavage rate of Co(II)(Chro)(2) (1.2 × 10(-3) s(-1)) is higher than those of Fe(II)(Chro)(2) and Ni(II)(Chro)(2), which were calculated to be 1 × 10(-4) and 3.1 × 10(-4) s(-1), respectively. Consistent with the SPR and UV melting results, Ni(II)(Chro)(2) possesses the highest inhibitory effect on in vitro transcription and c-myc transcription within cells compared to Co(II)(Chro)(2) and Fe(II)(Chro)(2). By comparing the cytotoxicity among Co(II)(Chro)(2), Fe(II)(Chro)(2), and Ni(II)(Chro)(2) to several cancer cell lines, our studies concluded that Ni(II)(Chro)(2) displayed more potential antitumor activities than Co(II)(Chro)(2) and Fe(II)(Chro)(2) did due to its higher DNA-acting efficacy. Changes to the divalent metal ions in the dimeric Chro complexes have been correlated with improved anticancer profiles. The availability of new metal derivatives of Chro may introduce new possibilities for exploiting the unique properties of this class of compounds for therapeutic applications.

Membrane-active peptides and the clustering of anionic lipids.

There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids.

Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation.

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.