Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.     

Flow and anchoring effects on nematic fluctuations in confined geometry

Light scattering observation of nematic director fluctuations in confined geometries can be used to obtain interaction parameters of liquid crystals with surfaces. We present the basics of the method and some examples of the results in planar and cylindrical geometries. These results were obtained after neglecting the coupling of the director motion to flow. We give analytical and numerical results of flow effects on director fluctuations in a slab. The backflow contribution to the effective viscosity is strongly suppressed so that the results for the anchoring energy remain valid. Modal dispersion relations show an interesting behaviour of avoiding crossings.     

Teaching liquid crystals – observation inspired curiosity

Soft matter has become involved in all aspects of everyday life over the past few decades, from diapers and the water-absorbing colloidal crystals hidden in them to the omnipresent liquid crystal displays. This article discusses an introduction to one example of soft matter – liquid crystals – at various educational levels. It stresses the importance of experimental work and presents a few simplified versions of elaborate techniques graduate students later meet in laboratories. A set of simple experiments, which illustrate typical phenomena for liquid crystals, but use different approaches or different materials than liquid crystals, are also presented. Drawing upon analogies is an essential part of an active researcher’s thought processes. By providing analogous experiences and showing clear analogies between various phenomena, a lecturer can train students in the use of analogies as a standard approach when encountering new problems.     

Removal of Flotation Collector O-Isopropyl-N-ethylthionocarbamate from Wastewater

Flotation collector O-isopropyl N-ethylthionocarbamate (IPETC) is widely used for separation of sulfide ores. Its removal from water by several oxidation processes was studied. Photocatalytic oxidation with air in the presence of iron salts, utilizing solar irradiation or artificial UV-A light is very efficient. Oxidation leads through the formation of O-isopropyl N-ethylcarbamate and several other reaction intermediates to total decomposition of organic compound in the final stage in 1 day. Similar results were obtained with a Fenton type oxidation with hydrogen peroxide and iron salts. Treatment with sodium hypochlorite yields mainly O-isopropyl N-ethylcarbamate. The formation of this compound in wastewaters can be of concern, since simple alkyl carbamates are cancer suspect agents.     

Agglutination of like-charged red blood cells induced by binding of beta2-glycoprotein I to outer cell surface

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.     

Novel inhibitors of trihydroxynaphthalene reductase with antifungal activity identified by ligand-based and structure-based virtual screening

Curvularia lunata is a dark pigmented fungus that is the causative agent of several diseases in plants and in both immunodeficient and immunocompetent patients. 1,8-Dihydroxynaphthalene-melanin is found in the cell wall of C. lunata and is believed to be the important virulence factor of dematiaceous fungi. Trihydroxynaphthalene reductase is an enzyme of the 1,8-dihydroxynaphthalene-melanin biosynthetic pathway, and it thus represents an emerging target for the development of novel fungicides and antimycotics. In the present study, we describe novel inhibitors of trihydroxynaphthalene reductase from C. lunata. These inhibitors were identified by ligand-based three-dimensional similarity searching and docking to a homology-built model and by subsequent biochemical and antifungal evaluation. Discovery of competitive inhibitors with K(i) values in low micromolar and even nanomolar concentration range proves the aplicability of homology-built model of 3HNR for hit finding by virtual screening methods.     

The effect of resting transmembrane voltage on cell electropermeabilization: a numerical analysis

The transmembrane voltage induced due to applied electric field superimposes to the resting transmembrane voltage of the cell. On the part of the cell membrane, where the transmembrane voltage exceeds the threshold transmembrane voltage, changes in the membrane occur, leading to increase in membrane permeability known as electropermeabilization. This part of the cell membrane represents the permeabilized area through which the transport of molecules occurs. In this paper we calculated numerically the permeabilized area for different electric field strength, resting transmembrane voltage, cell shape and cell orientation with respect to the applied electric field. Results show that when the transmembrane voltage is near the threshold transmembrane voltage, the permeabilized area of the cell is increased on the anodic side and decreased on the cathodic side due to the resting transmembrane voltage. In some cases, only anodic side of the cell is permeabilized. Therefore, by using bipolar pulses, the permeabilized area can be significantly increased and consequentially also the efficiency of electropermeabilization. However, when the induced transmembrane voltage is far above the threshold, the effect of the resting transmembrane voltage is negligible. These observations are valid for different cell shapes and orientations.