Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermodule: spectroscopic/functional individuality of plasminogen kringle domains

The plasminogen kringle 2 (K2HPg) and kringle 3 (K3HPg) modules occur in tandem within the polypeptide segment that affords the heavy chain of plasmin. The K2HPg and K3HPg are unique among the plasminogen kringle domains in that they also are linked to each other via the Cys169-Cys297 (Cys4 of K2HPg to Cys43 of K3HPg, kringle numbering convention) disulfide bridge, thus generating a K2HPg-K3HPg "supermodule". The kringle (2 + 3) sequence of human plasminogen (r-EE[K2HPgK3HPg]DS) was expressed in Escherichia coli, using an expression vector containing the phage T5 promoter/operator N250PSN250P29 and the codons for an N-terminal hexahistidine tag to ensure the isolation of the recombinant protein by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose under denaturing and reducing conditions. Kringle (2 + 3) was refolded in the presence of glutathione redox buffer. By taking advantage of the lysine affinity of kringle 2, the protein was purified by affinity chromatography on lysine-Bio-Gel. Recombinant kringle (2 + 3) was identified by amino acid composition, N-terminal sequence and mass determination. The 1H NMR spectrum shows that the intact r-K2HPgK3HPg is properly folded. By reference to spectra of the individual kringles, r-K2HPg and r-K3HPg, resonances of the K2HPg and K3HPg components in the spectrum of the intact r-K2HPgK3HPg can be readily distinguished. The strictly conserved Leu46 residue (kringle residue number convention) yields delta-methyl signals that are characteristic for K2HPg and K3HPg, exhibiting chemical shifts of -0.87 and -0.94 ppm, respectively, which are distinct from those of K1HPg, K4HPg, and K5HPg, (-1.04 to -1.05 ppm). Thus, the high-field Leu46 signals from K2HPg and K3HPg are well resolved from those of other kringles and can be identified unambiguously in spectra of the K1HPgK2HPgK3HPg elastolytic fragment of plasminogen as well as in spectra of Glu-plasminogen. Overall, r-K2HPgK3HPg exhibits broader resonance line widths than does the K1HPg component, consistent with a lesser mobility of the K2HPgK3HPg segment within the K1HPgK2HPgK3HPg fragment, a reflection of the extra structural constraint imposed by the disulfide bridge linking K2HPg to K3HPg. The ligand 6-aminohexanoic acid (6-AHA), which is known to interact with r-K2HPg but not with r-K3HPg, selectively perturbs K2 aromatic signals in the intact r-K2HPgK3HPg spectrum while leaving K3 resonances largely unaffected. Association constant (K(a)) values for 6-AHA determined from 1H NMR ligand titration experiments yield K(a) approximately 2.2 +/- 0.3 mM(-1) for the intact r-K2HPgK3HPg, comparable to K(a) approximately 2.3 +/- 0.2 mM(-1) determined for the isolated r-K2HPg, which demonstrates that the interactions of 6-AHA with the K2HPg ligand-binding site are not significantly affected by the neighboring K3HPg domain within the intact r-K2HPgK3HPg supermodule.     

The Mechanisms through Which Certain Variables Influence Customer Loyalty: The Mediating Roles of Perceived Value and Satisfaction

This study surveyed 123 bank users who experienced problems with their banks’ online banking services, with the aim of understanding the mechanisms through which certain variables influence customer loyalty. Principally, the possible mediating roles of value and satisfaction on the relationships between recovery and loyalty and between ISO 9001 and loyalty were scrutinized. The dimensionalities of the scales were assessed using exploratory and confirmatory factor analysis. Thereafter, structural equation modeling and multiregression analyses were used to test the proposed model. The overall results showed that service recovery can be used to enhance customers’ satisfaction and perception of value and, therefore, customer loyalty. This study also confirmed the partial mediating roles of customers’ satisfaction and perception of value in the relationship between service recovery and loyalty. In contrast, ISO 9001 has no influence on perceived value, satisfaction, and loyalty; the mediating roles of both perceived value and satisfaction were not supported in the relationship between ISO 9001 and loyalty. In practice, even though ISO 9001 appeared to offer few extras to satisfy and retain customers, its use is still advisable because of the other potential benefits that it provides.     

Experience over time: evaluating the experience of use of a squeezable interface in the medium term

The paper presents the user experience evaluation of Squeeze Me, an interactive cover for tablet and smartphone that enables continuous and expressive interaction with electronic devices. The cover has been used to implement ”Squeeze to zoom”, a mobile application to zoom in and out while taking a photograph from a tablet. The experience of use was evaluated in the short and medium term, comparing the Squeeze Me interaction modality with classic modalities for zooming in and out commonly available on tablets and smartphones. The evaluation process was conducted using AttrakDiff [3] a questionnaire that measures hedonic stimulation and identity, as well as pragmatic qualities and attractiveness of software products. Participants were asked to try out different interaction modalities for comparison in the short-term (67 people) and over 4 weeks (8 people). Results obtained in the short-term evaluation reveal that “Squeeze to zoom” was awarded higher values than the classic “Slide to zoom” in the hedonic quality-stimulation and attractiveness dimensions, whilst it obtained lower values in the pragmatic quality and hedonic quality-identity. However, the experience of use changed over time. During the longitudinal study, the usability of “Squeeze to zoom” improved whilst the attractiveness of “Slide to zoom” decreases significantly. Furthermore results reveal that “Squeeze to zoom” is significantly more appreciated for its hedonic qualities and the effect is maintained over time. This study highlights the importance of evaluating the experience of use over time, a practice that is almost ignored in the literature on Experience Design.     

Tube leukocyte adherence inhibition assay for the detection of anti-tumour immunity. II. Monocyte reacts with tumour antigen via cytophilic anti-tumour antibody

The peripheral blood monocyte is the reactive cell in the tube LAI assay. The monocyte loses its properties of adherence to glass upon exposure to specific antigen. Two different experiments to determine if lymphocytes, when they reacted with tumour, released mediators that were responsible for inhibiting monocyte glass adherence, gave negative results. The mechanism wherby the specific tumour antigen appeared to be recognized was the binding of cytophilic IgG antitumour antibody to receptors on the cell surface of the monocyte. The results of the experiments indicate that normal peripheral blood monocytes could be made specifically reactive ("armed") to the tumour extract by incubating normal peripheral blood leukocytes with serum from a reactive cancer patient. IgG isolated from "arming" sera was shown to have the capacity to sensitize normal leukocytes. Patients with breast cancer or malignant melanoma with limited tumour burdens had free cytophilic anti-tumour antibody in their serum, whereas the serum of patients with large tumour burdens (metastatic cancer), whose leukocytes did not react in the tube LAI assay, did not "arm".     

Low-Temperature Synthesis and Properties of LaMnO3±d and La0.67R0.33MnO3±d (R = Ca, Sr, Ba) from Citrate Precursors

LaMn_{1− y} ³⁺Mn _y ⁴⁺O_3±d and La_0.67R_0.33Mn_{1− y} ³⁺Mn _y ⁴⁺O_3±d (R = Ca, Sr, Ba) phases were synthesized at 350°C by using very reactive, amorphous precursors obtained from the stoichiometric citrate solutions. The chemical process was optimized with respect to the solution concentration, pH, and additives. The precursor reactions were investigated as a function of the cation stoichiometry and the additive by simultaneous thermal and thermogravimetric analysis and X-ray diffraction. The reaction pathway was found to be independent of the cation stoichiometry, but related to the acid or base additive. The annealing temperature was systematically increased in the 350–1200°C interval and the La_0.67Sr_0.33MnO_3±d properties (i.e., crystal sizes, Mn average valence, Curie temperature, magnetization, magnetic susceptibility) were measured and found to vary consistently as a function of it.     

Influence of the overlap parameter on the convergence of the ptychographical iterative engine

The ptychographical iterative engine (PIE) algorithm is examined with both simulated and experimental scanning coherent-diffraction microscopy data. The optimum overlap in terms of image quality, dose on the sample and time of measurements is determined using simulated diffraction data. The validity of the results is supported by experimental helium-neon laser light diffraction data.     

X-ray microscopy of plant cells by using LiF crystal as a detector

A lithium fluoride (LiF) crystal has been utilized as a new soft X-ray detector to image different biological samples at a high spatial resolution. This new type of image detector for X-ray microscopy has many interesting properties: high resolution (nanometer scale), permanent storage of images, the ability to clear the image and reuse the LiF crystal, and high contrast with greater dynamic range. Cells of the unicellular green algae Chlamydomonas dysosmos and Chlorella sorokiniana, and pollen grains of Olea europea have been used as biological materials for imaging. The biological samples were imaged on LiF crystals by using the soft X-ray contact microscopy and contact micro-radiography techniques. The laser plasma soft X-ray source was generated using a Nd:YAG/Glass laser focused on a solid target. The X-ray energy range for image acquisition was in the water-window spectral range for single shot contact microscopy of very thin biological samples (single cells) and around 1 keV for multishots microradiography. The main aim of this article is to highlight the possibility of using a LiF crystal as a detector for the biological imaging using soft X-ray radiation and to demonstrate its ability to visualize the microstructure within living cells.     

Reduced Environmental Dose Rates Are Responsible for the Increased Susceptibility to Radiation-Induced DNA Damage in Larval Neuroblasts of Drosophila Grown inside the LNGS Underground Laboratory

A large amount of evidence from radiobiology studies carried out in Deep Underground Laboratories support the view that environmental radiation may trigger biological mechanisms that enable both simple and complex organisms to cope with genotoxic stress. In line with this, here we show that the reduced radiation background of the LNGS underground laboratory renders Drosophila neuroblasts more sensitive to ionizing radiation-induced (but not to spontaneous) DNA breaks compared to fruit flies kept at the external reference laboratory. Interestingly, we demonstrate that the ionizing radiation sensitivity of flies kept at the LNGS underground laboratory is rescued by increasing the underground gamma dose rate to levels comparable to the low-LET reference one. This finding provides the first direct evidence that the modulation of the DNA damage response in a complex multicellular organism is indeed dependent on the environmental dose rate.     

Genotoxicological Characterization of (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR and Their Association

The novel psychoactive substance (NPS) 4-Methyl-5-(4-methylphenyl)-4,5-dihydroxazol-2-amine (4,4′-DMAR) shows psychostimulant activity. Data on the acute toxicity of 4,4′-DMAR are becoming increasingly available, yet the long-term effects are still almost unknown. In particular, no data on genotoxicity are available. Therefore, the aim of the present study was to evaluate its genotoxic potential using the “In Vitro Mammalian Cell Micronucleus Test” (MNvit) on (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR and their associations. The analyses were conducted in vitro on human TK6 cells. To select suitable concentrations for MNvit, we preliminarily evaluated cytotoxicity and apoptosis. All endpoints were analysed by flow cytometry. The results reveal the two racemates’ opposite behaviours: (±)cis-4,4′-DMAR shows a statistically significant increase in micronuclei (MNi) frequency that (±)trans-4,4′-DMAR is completely incapable of. This contrast confirms the well-known possibility of observing opposite biological effects of the cis- and trans- isomers of a compound, and it highlights the importance of testing single NPSs that show even small differences in structure or conformation. The genotoxic capacity demonstrated stresses an additional alarming toxicological concern related to this NPS. Moreover, the co-treatments indicate that consuming both racemates will magnify the genotoxic effect, an aspect to consider given the unpredictability of illicit drug composition.     

Drought Stress Pre-Treatment Triggers Thermotolerance Acquisition in Durum Wheat

Durum wheat is strongly affected by climatic constraints such as high temperatures and drought, which frequently lead to yield reduction. Damages due to high temperatures are related to plant thermotolerance, a trait determined by two components: basal and acquired thermotolerance. In this study, the effect of drought and heat stress imposed singularly or sequentially was investigated in ten durum wheat cultivars (cvs) at the physiological and molecular level. The traits analyzed were cell membrane stability, relative water content, proline content, and expression level of several genes for heat shock proteins (HSPs). Our results indicate that drought priming can induce the acquisition of thermotolerance in most cultivars already classified as able to acquire thermotolerance by heat pre-treatment. Proline accumulation was correlated to cell membrane stability, meaning that the most thermotolerant cvs were able to accumulate higher levels of proline. Acquired thermotolerance is also due to the activation of HSP gene expression; similarly, pre-treatment with water stress was able to activate HSPs expression. The results reported indicate that water stress plays an important role in inducing thermotolerance, comparable to mild heat stress pre-treatment. This is the first report on the effect of drought stress on the acquisition of thermotolerance.