Analytical Ultracentrifugation as a Matrix-Free Probe for the Study of Kinase Related Cellular and Bacterial Membrane Proteins and Glycans

Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free—providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review—designed for the non-hydrodynamic expert—examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system—studied in aqueous and detergent based solvent systems—and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.

Microbiological evaluation of different antiseptic povidone-iodine and chlorhexidine formulations after intentional contamination of containers

This study aimed to evaluate the survival rate of microorganisms within different antiseptic formulations - povidone-iodine (PVP-I) and chlorhexidine (CHX) - after intentional contamination, and to establish the minimum care necessary to ensure sterilization of non-disposable antiseptic solution containers. A laboratory study was performed with 180 antiseptic containers, which were contaminated with Serratia marcescens [1 x 105 UFC/mL]. The containers were closed and stored, at room temperature, during seven days and shaken daily. The antiseptic cultures were evaluated to be 100% negative to Serratia marcescens in all of the non-disposable containers. These results suggested that antiseptic solutions inactivate microorganisms [1 x 105 UFC/mL]. Since cleaned antiseptic containers have around 102 UFC coming from tap water, it can be inferred that cleansing is a safe minimum procedure to ensure reuse of containers for distribution of CHX and PVP-I solutions in aqueous, detergent and alcoholic formulations.

Identification of Serine Proteases from Leishmania braziliensis

Leishmania (V.) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nα-benzoyl-l-arginine ethyl ester hydrochloride and Nα-p-tosyl-l-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.

Intrinsic near Infrared Sensor for the Determination of the Drainage Behaviour of Aqueous Detergent Solutions

Surfactants, detergents or tensides are synonyms for substances that, even at low concentrations, can drastically alter the interfacial properties of a multiphase system. Their main application fields—among many others—are cleaning processes in households, trade and industry. A certain substance-specific concentration (c.m.c.) is generally considered to be optimal for these applications. However, the fast and inexpensive determination of tenside concentrations is still a difficult task. Our approach to this problem is based on the fact that the draining behaviour of aqueous solutions on hard surfaces depends strongly on the actual concentration of detergents. Near the critical micelle concentration a stable fluid film remains on the wetted surface. The use of optic-intrinsic sensors in the NIR allows the detection of these changes in a straightforward manner by monitoring the sensor response after retrieving the sensor from the sample liquid. A potential application of this sensor principle is expected for controlling the detergent addition mainly in large-scale cleaning devices. This would be of both economical and ecological advantage.