Studies on the secondary structure of nuclear ribonucleic acids

Rat liver nuclei were separated into two fractions, the nucleolus and the nucleoplasm, by a combined salt-enzymic extraction. RNA was purified from both sources and analysed on sucrose density gradients. In both fractions a prominent heterogeneous RNA class with mean sedimentation coefficient of 18S was found. This material was analysed by measuring the rate of reaction with formaldehyde, the ultraviolet absorbance-temperature profile, the spectrophotometric observation of conformational changes as a function of pH, the spectrophotometric titration of uracil and guanine residues and the effect of both temperature and ionic strength on the spectrophotometric titration of cytosine residues. Nucleoplasmic 18S RNA fraction exhibited, on heating and also by adjustment of the pH to 2.5, a hyperchromicity of about 16%, close to that observed, in control experiments, for ribosomal RNA (22%). Titration of cytosine residues in solutions containing 1mm-NaCl and 0.1m-NaCl yielded pK values equal to 4.41 and 3.84 respectively. These results suggest that this RNA fraction is composed of structurally complex polymers. The hypochromicity of the nucleolar 18S RNA fraction determined by heating or adjusting the pH to 2.5, was not greater than 6% of the initial value. The rate of reaction with formaldehyde was 88% of that observed for the hydrolysed 18S fraction which suggested only 12% hydrogen bonding. pK values for uracil and guanine residues were 10.1 and 10.05 respectively. Titration of cytosine residues yielded a pK of 4.10, which was found to be independent of temperature and ionic strength variations.     

Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.     

Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Water cytotoxicity and dioxins bioaccumulation in an Egyptian delta wetland ecosystem

Manzala Lake, as one of the main Egyptian wetland ecosystems, is facing risks of pollution. An in vitro cytotoxicity test using a mammalian cell line was employed to determine the toxicity of multiple pollutants in the water and Tilapia zillii fish sampled from the lake. The concentrations of seven polychlorinated dibenzo-p-dioxins and ten polychlorinated dibenzofurans were investigated in water and muscle of the fish in 2014. Cytotoxicity testing showed that the percentage inhibition of cell viability in the studied sites ranged between 56.16% and 83.22%. Dioxin analysis indicated that the average concentrations of 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzofuran and 1,2,3,4,6,7,8,9-octachlorodibenzofuran were higher than the toxic equivalence quotients (TEQs) set by the World Health Organization (WHO) in all water and fish muscle samples; however, the average concentration of 2,3,7,8-tetrachlorodibenzofuran was higher only in fish muscle samples. The bioaccumulation factor (BAF) ranged dramatically between 2 and 58.5 for the detected dioxins. Adverse human health effects through the consumption of fish are not expected, because dioxin levels in fish muscle are deemed safe for human consumption. Implementation of a strategic multidisciplinary action plan is strongly recommended to sustain this delta wetland ecosystem.