Determination of the structural integrity of DNA from pathogenic enterobacteria

Among the enterobacterial strains under study, more organisms in the stationary phase of growth have been found to have nicks in their DNA than those in the exponential phase. Bacteria less sensitive to ultraviolet irradiation have the least number of nicks in each phase of growth. The number of nicks in different strains belonging to the serovar is sufficiently stable. Virulent and avirulent forms show no difference in this characteristic.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Synthesis of a new carrier for immunization: polytuftsin. Two examples of its use with peptides selected in the hepatitis B surface antigen

Sequential poly(Arg-Thr-Lys-Pro) consisting mainly of the repeat of tuftsin Thr-Lys-Pro-Arg was synthesized by condensing the p-nitrophenyl ester of Arg(HCl)-Thr-Lys-(2-Cl-Z)-Pro in the presence of HOBt. Two haptenic sequences of the Pre-S region of hepatitis B virus antigen (10-26 and 39-55) were prepared by solid phase and coupled to polytuftsin via glutaraldehyde. The peptides, either free or coupled to polytuftsin, were administrated to mice and the antisera were assayed by ELISA. Coupling the peptides to the polypeptide significantly improved the anti-peptide antibody titer in Freund complete adjuvant or in NaCl 0.9%. Cross-reaction between antibodies induced by the peptides and the native protein was also improved. Polytuftsin alone is very poorly immunogenic.     

State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Thermal analysis of the dentine tubule under hot and cold stimuli using fluid–structure interaction simulation

The objective of this study is to compare the thermal stress changes in the tooth microstructures and the hydrodynamic changes of the dental fluid under hot and cold stimuli. The dimension of the microstructures of eleven cats’ teeth was measured by scanning electron microscopy, and the changes in thermal stress during cold and hot stimulation were calculated by 3D fluid–structure interaction modeling. Evaluation of results, following data validation, indicated that the maximum velocities in cold and hot stimuli were ??410.2?±?17.6 and +?205.1?±?8.7 µm/s, respectively. The corresponding data for maximum thermal stress were ??20.27?±?0.79 and +?10.13?±?0.24 cmHg, respectively. The thermal stress caused by cold stimulus could influence almost 2.9 times faster than that caused by hot stimulus, and the durability of the thermal stress caused by hot stimulus was 71% greater than that by cold stimulus under similar conditions. The maximum stress was on the tip of the odontoblast, while the stress in lateral walls of the odontoblast and terminal fibril was very weak. There is hence a higher possibility of pain transmission with activation of stress-sensitive ion channels at the tip of the odontoblast. The maximum thermal stress resulted from the cold stimulus is double that produced by the hot stimulus. There is a higher possibility of pain transmission in the lateral walls of the odontoblast and terminal fibril by releasing mediators during the cold stimulation than the hot stimulation. These two reasons can be associated with a greater pain sensation due to intake of cold liquids.     

Effects of NaCl and iso-osmotic PEG stress on growth,osmolytes accumulation and antioxidant defense in cultured sugarcane cells

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.