Purine 5′,8-cyclo-2′-deoxynucleoside lesions: formation by radical stress and repair in human breast epithelial cancer cells

5′,8-Cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) in their two diastereomeric forms, 5′S and 5′R, are tandem lesions produced by the attack of hydroxyl radicals to the purine moieties of DNA. Their formation has been found to challenge the cells’ repair machinery, initiating the nucleotide excision repair (NER) for restoring the genome integrity. The involvement of oxidatively induced DNA damage in carcinogenesis and the reduced capacity of some cancer cell lines to repair oxidised DNA base lesions, intrigued us to investigate the implication of these lesions in breast cancer, the most frequently occurring cancer in women. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we measured the levels of diastereomeric cdA’s and cdG’s in estrogen receptor-alpha positive (ER-α) MCF-7 and triple negative MDA-MB-231 breast cancer cell lines before and after exposure to two different conditions: ionising radiations and hydrogen peroxide, followed by an interval period to allow DNA repair. An increase at the measured levels of all four lesions, i.e. 5′S-cdA, 5′R-cdA, 5′S-cdG and 5′R-cdG, was observed either after γ-irradiation (5?Gy dose) or hydrogen peroxide treatment (300?μM) compared to the untreated cells (control), independently from the length of the interval period for repair. For comparison reasons, we also measured the levels of 8-oxo-2′-deoxyadenosine (8-oxo-dA), a well-known oxidatively induced DNA damage lesion and base excision repair (BER) substrate. The collected data indicate that MCF-7 and MDA-MB-231 breast cancer cells are highly susceptible to radiation-induced DNA damage, being mainly defective in the repair of these lesions.     

Interactions of the dipeptide paralysin beta-Ala-Tyr and the aminoacid Glu with phospholipid bilayers

Existing evidence points out that the biological activity of beta-Ala-Tyr may in part related to its interactions with the cell membranes. For comparative reasons the effects of Glu were also examined using identical techniques and conditions. In order to examine their thermal and dynamic effects on membrane bilayers a combination of DSC, Raman and solid state NMR spectroscopy on DPPC/water model membranes were applied and the results were compared. DSC data showed that Glu perturbs to a greater degree the model membrane compared to beta-Ala-Tyr. Thus, alteration of the phase transition temperature and half width of the peaks, abolishment of the pretransition and influence on the enthalpy of the phase transition were more pronounced in the Glu loaded bilayers. Raman spectroscopy showed that incorporation of Glu in DPPC/water bilayers increased the order in the bilayers in contrast to the effect of the dipeptide. Several structural and dynamical properties of the DPPC multilamellar bilayers with and without the dipeptide or Glu were compared using high resolution C-13 MAS (Magic Angle Spinning) spectra and spectral simulations of inhomogeneously broadened, stationary P-31 NMR lineshapes measured under CP (Cross-polarization) conditions. These methods revealed that the aminoacid Glu binds in the close realm of the phosphate in the hydrophilic headgroup of DPPC while beta-Ala-Tyr is located more deeply inside the hydrophobic zone of the bilayer. The P-31 NMR simulations indicated restricted fast rotary motion of the phospholipids about their long axes in the organized bilayer structure. Finally, by the applied methodologies it is concluded that the two molecules under study exert dissimilar thermal and dynamic effects on lipid bilayers, the Glu improving significantly the packing of the lipids in contrast to the smaller and opposite effect of the dipeptide.     

Inflammation,Endothelial Dysfunction and Increased Left Ventricular Mass in Chronic Kidney Disease (CKD) Patients: A Longitudinal Study

Introduction

Within this longitudinal study we investigated the association of inflammation markers C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) and endothelial dysfunction markers intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) with left ventricular mass indexed for height²·⁷¹ (LVMI) in hypertensive predialysis CKD patients.

Material and Methods

From 2004 to 2005, 182 incident consecutive adult patients from the outpatient CKD clinics of two hospitals in Greece with CKD and hypertension or using antihypertensive medication, were included. Of these, 107 patients underwent CRP (mg/l) and LVMI (g/height²·⁷¹) measurements annually for three years.

Results

In the longitudinal analyses, using linear mixed modeling, a higher IL-6 (ß = 1.9 (95%ci:0.38;3.5), inflammation score based on CRP, IL-6 and TNF-α (ß = 5.0 (95%ci:0.72; 9.4) and VCAM-1 (ß = 0.01 (95%ci:0.005;0.02) were associated with higher LVMI. These models were adjusted for age, gender and primary renal disease, and for confounders that on top changed the beta with ≥10%, i.e. diuretic use (for IL-6 and inflammation score).

Conclusion

The results suggest that in predialysis CKD patients, inflammation as well as endothelial dysfunction may play an important role towards the increase in LVMI.     

Spray-dried voriconazole–cyclodextrin complexes: Solubility,dissolution rate and chemical stability

The present work investigates the effect of complexation with hydroxypropyl-beta-cyclodextrin (HPBCD) and 2-O-methyl-beta-cyclodextrin (2-O-MBCD), on voriconazole solubility, dissolution rate and chemical stability. Drug–cyclodextrin complexes were prepared as aqueous solutions, which were spray-dried, and their properties were compared to wet ground samples and physical mixtures. DSC analysis revealed absence of crystalline voriconazole from spray-dried complexes. FTIR spectroscopy indicated changes in the H-bonding network of the hydroxyl groups of cyclodextrin following drug inclusion. Dissolution rate of voriconazole was significantly higher from spray-dried complexes with either cyclodextrin in comparison with free drug, physical mixtures, or wet ground mixtures. However, two degradation impurities were found in aged samples, with slightly higher impurity level with HPBCD. Performed solubility studies suggested that 2-O-MBCD is more efficient solubilizer. Molecular docking simulations showed a difference in the 1:1 binding affinities and sites, with HPBCD surprisingly forming complexes of much lower energy, thus suggesting a multiple rather than a 1:1 complexation.     

LDL receptor deficiency or apoE mutations prevent remnant clearance and induce hypertriglyceridemia in mice

We have used adenovirus-mediated gene transfer and bolus injection of purified apolipoprotein E (apoE) in mice to determine the contribution of LDL receptor family members in the clearance of apoE-containing lipoproteins in vivo and the factors that trigger hypertriglyceridemia. A low dose [5 x 10(8) plaque-forming units (pfu)] of an adenovirus expressing apoE4 did not normalize plasma cholesterol levels of apolipoprotein E-deficient (apoE(-/-)) x low density lipoprotein receptor-deficient (LDLr(-/-)) mice and induced hypertriglyceridemia. A similar phenotype of combined dyslipidemia was induced in apoE(-/-) or apoE(-/-) x LDLr(-/-) mice after infection with a low dose (4 x 10(8) pfu) of an adenovirus expressing the apoE4[R142V/R145V] mutant previously shown to be defective in receptor binding. In contrast, a low dose of 5 x 10(8) pfu of the apoE4-expressing adenovirus corrected hypercholesterolemia in apoE(-/-) mice and did not trigger hypertriglyceridemia. Bolus injection of purified apoE in apoE(-/-) x LDLr(-/-) mice did not clear plasma cholesterol levels and induced mild hypertriglyceridemia. In contrast, similar injection of apoE in apoE(-/-) mice cleared plasma cholesterol and caused transiently mild hypertriglyceridemia. These findings suggest that a) the LDL receptor alone can account for the clearance of apoE-containing lipoproteins in mice, and the contribution of other receptors is minimal, and b) defects in either the LDL receptor or in apoE that affect its interactions with the LDL receptor, increase the sensitivity to apoE-induced hypertriglyceridemia in mice.     

An eutomer/distomer ratio near unity does not justify non-enantiospecific assay methods in bioequivalence studies

The aim of the present investigation was to compare the pharmacokinetics of two tablet formulations of 600 mg of racemic ibuprofen obtained using enantiospecific and non-enantiospecific assays, in order to explore if chiral assays should be employed in bioequivalence studies of chiral active substances. The stereoselective assay showed that, for both formulations, there was an initial phase where (R)-ibuprofen was the predominant enantiomer followed by a final phase where (S)-ibuprofen was the predominant one. Results from both analytical methods proved that the two formulations were bioequivalent. However, the chiral bioanalytical method detected a larger difference in the eutomer than that showed by the nonchiral bioanalytical method. In conclusion, although the exposure ratios of enantiomers are near unity, the measurement of unresolved ibuprofen alone is not an adequate measure of bioequivalence since it may mask the actual difference in the eutomer exposure among formulations.     

Acyclic peptides incorporating the d‐Phe‐2‐Abz turn motif: Investigations on antimicrobial activity and propensity to adopt β‐hairpin conformations

Three linear peptides incorporating d ‐Phe‐2‐Abz as the turn motif are reported. Peptide 1 , a hydrophobic β‐hairpin, served as a proof of principle for the design strategy with both NMR and CD spectra strongly suggesting a β‐hairpin conformation. Peptides 2 and 3, designed as amphipathic antimicrobials, exhibited broad spectrum antimicrobial activity, with potency in the nanomolar range against Staphylococcus aureus. Both compounds possess a high degree of selectivity, proving non‐haemolytic at concentrations 500 to 800 times higher than their respective minimal inhibitory concentrations (MICs) against S. aureus. Peptide 2 induced cell membrane and cell wall disintegration in both S. aureus and Pseudomonas aeruginosa as observed by transmission electron microscopy. Peptide 2 also demonstrated moderate antifungal activity against Candida albicans with an MIC of 50 μM. Synergism was observed with sub‐MIC levels of amphotericin B (AmB), leading to nanomolar MICs against C. albicans for peptide 2 . Based on circular dichroism spectra, both peptides 2 and 3 appear to exist as a mixture of conformers with the β‐hairpin as a minor conformer in aqueous solution, and a slight increase in hairpin population in 50% trifluoroethanol, which was more pronounced for peptide 3 . NMR spectra of peptide 2 in a 1:1 CD₃CN/H₂O mixture and 30 mM deuterated sodium dodecyl sulfate showed evidence of an extended backbone conformation of the β‐strand residues. However, inter‐strand rotating frame Overhauser effects (ROE) could not be detected and a loosely defined divergent hairpin structure resulted from ROE structure calculation in CD₃CN/H₂O. The loosely defined hairpin conformation is most likely a result of the electrostatic repulsions between cationic strand residues which also probably contribute towards maintaining low haemolytic activity.     

Negative Purifying Selection Drives Prion and Doppel Protein Evolution

The prion protein (PrP) when misfolded into the pathogenic conformer PrP^Sc is the major causative agent of several lethal transmissible spongiform encephalopathies in mammals. Studies of evolutionary pressure on the corresponding gene using different datasets have yielded conflicting results. In addition, putative PrP or PrP interacting partners with strong similarity to PrP such as the doppel protein have not been examined to determine if the same evolutionary mechanisms apply to prion paralogs or if there are coselected sites that might indicate how and where the proteins interact. We examined several taxonomic groups that contain model organisms of prion diseases focusing on primates, bovids, and an expanded dataset of rodents for selection pressure on the prion gene (PRNP) and doppel gene (PRND) individually and for coevolving sites within. Overall, the results clearly indicate that both proteins are under strong selective constraints with relaxed selection on amino acid residues connecting α-helices 1 and 2.