Calmodulin-mediated reversible immobilization of enzymes

This work demonstrates the use of the protein calmodulin, CaM, as an affinity tag for the reversible immobilization of enzymes on surfaces. Our strategy takes advantage of the of the reversible, calcium-mediated binding of CaM to its ligand phenothiazine and of the ability to produce fusion proteins between CaM and a variety of enzymes to reversibly immobilize enzymes in an oriented fashion to different surfaces. Specifically, we employed two different enzymes, organophosphorus hydrolase (OPH) and beta-lactamase and two different solid supports, a silica surface and cellulose membrane modified by covalently attaching a phenothiazine ligand, to demonstrate the versatility of our immobilization method. Fusion proteins between CaM-OPH and CaM-beta-lactamase were prepared by using genetic engineering strategies to introduce the calmodulin tail at the N-terminus of each of the two enzymes. In the presence of Ca(2+), CaM adopts a conformation that favors interaction between hydrophobic pockets in CaM and phenothiazine, while in the presence of a Ca(2+)-chelating agent such as EGTA, the interaction between CaM and phenothiazine is disrupted, thus allowing for removal of the CaM-fusion protein from the surface under mild conditions. CaM also acts as a spacer molecule, orienting the enzyme away from the surface and toward the solution, which minimizes enzyme interactions with the immobilization surface. Since the method is based on the highly selective binding of CaM to its phenothiazine ligand, and this is covalently immobilized on the surface, the method does not suffer from ligand leaching nor from interference from other proteins present in the cell extract. An additional advantage lies in that the support can be regenerated by passing through EGTA, and then reused for the immobilization of the same or, if desired, a different enzyme. Using a fusion protein approach for immobilization purposes avoids the use of harsh conditions in the immobilization and/or regeneration steps, which could cause inactivation of the immobilized enzyme. Moreover, we have demonstrated that the CaM affinity tag allows immobilization of enzymes on a variety of surfaces without compromising their enzymatic activity substantially; for example, the immobilized OPH retained more than 80% of the activity of the free enzyme. Our results with beta-lactamase showed the feasibility of using a phenothiazine surface in several consecutive loading and regeneration cycles. This can be advantageous when expensive and/or difficult to obtain immobilization surfaces have to be employed; the immobilization surface could be reused to immobilize the same or a different enzyme using the CaM affinity tail. We also determined that the phenothiazine-modified silica particles are stable for long periods of time, i.e., up to 2 years when stored at 4 degrees C. It is envisioned that this type of reversible immobilization may find applications in the development of reversible, reusable biosensors and bioreactors endowed with the additional advantage that the biological element at the surface of the sensor or bioreactor could be replaced under mild conditions when needed to sense or process a different target molecule.     

The Onset of Systemic Oxidative Stress Associated with the Accumulation of Lipid Peroxidation Product Acrolein in the Skin of Patients with Small-Vessel Vasculitis

Small-vessel vasculitis (SVV) is the inflammation of the vessel wall that can result in hemorrhage and/or ischemia. Among the histological findings in SVV are increased infiltrating neutrophils, which, due to their oxidative burst and myeloperoxidase activity, release excessive reactive oxygen species, triggering a chain reaction of lipid peroxidation and yielding reactive aldehydes such as acrolein. The implication of oxidative stress in the pathogenesis of SVV was studied, focusing on acrolein immunohistochemistry in the affected skin vessels and systemic stress response. Samples from SVV patients and healthy subjects were collected and analyzed for total serum peroxides, total antioxidant capacity, inflammatory and immunological parameters, as well as for the presence of acrolein–protein adducts in the skin tissue specimens. The obtained data showed that systemic redox homeostasis and iron metabolism are altered in SVV patients. Possible biomarkers in the evaluation of oxidative status, disease activity and prevalence were indicated. Furthermore, a strong correlation between the accumulation of acrolein–protein adducts in the skin and the progression of the disease was revealed. Thus, the results of this study demonstrate that SVV is not only associated with systemic oxidative stress but also with tissue-specific oxidative stress that promotes acrolein formation and protein modification correlating with the severity of cutaneous vasculitis.     

Competing processes in the photochemistry of picolines and their <Emphasis Type="Italic">N</Emphasis>-methyl salts: photoinduced charge transfer,phototransposition and photohydration

Photochemical reactivity of a series of picolines and their N-methylated salts has been investigated by preparative irradiations and UV–vis spectroscopy. Understanding competing photochemical processes and knowledge of their relative efficiencies is important in the application of pyridines as photocages or in the synthesis of complex polycycles. Contrary to previous reports for the gas phase, picolines are not reactive in the phototransposition, presumably due to protonation of the pyridine nitrogen in the excited state. Deuterium exchange was observed upon irradiation in CD₃CN–D₂O, but it was rationalized by photoionization and radical formation. On the other hand, N-methylated picoline salts are not protonated upon excitation. They undergo photohydration and phototransposition (Φ _R = 0.01–0.06). Upon irradiation of iodides, azabicyclic [3.1.0] hydration products were obtained. A difference in product distribution was observed between iodides and perchlorates, due to photoelimination of perchloric acid leading to the thermal aziridine ring opening. Moreover, excitation of iodide derivatives gives rise to change transfer transition forming iodide radicals that eventually give I₃ ^? with the quantum efficiency Φ _R = 0.015–0.02.     

Tunable Bidirectional Electroosmotic Flow for Diffusion-Based Separations

We present a new concept for on-chip separation that leverages bidirectional flow, to tune the dispersion regime of molecules and particles. The system can be configured so that low diffusivity species experience a ballistic transport regime and are advected through the chamber, whereas high diffusivity species experience a diffusion dominated regime with zero average velocity and are retained in the chamber. We detail the means of achieving bidirectional electroosmotic flow using an array of alternating current (AC) field-effect electrodes, experimentally demonstrate the separation of particles and antibodies from dyes, and present a theoretical analysis of the system, providing engineering guidelines for its design and operation.     

Pulsed corona discharge driven by Marx generator: Diagnostics and optimization for NOx treatment

A compact, repetitive Marx generator with an external trigger is constructed and coupled with a wire-to-plate corona reactor for a positive pulsed corona discharge studies. The reactor resistance and capacitance behavior during the pulse was observed. It was found that the reactor's capacitance increases three times during the pulse due to the streamer propagation from anode to grounded electrode. Using the time development of the capacitance and resistance during the pulse and the reactor inter-electrode distance, the streamer velocity has been calculated to be 1 × 10⁶ m/s, for system arrangement presented in this work. As an indicator of chemical activity of pulsed corona, ozone production was measured. Emission spectroscopy measurements in the UV region were performed to detect species that appear in the discharge and to determine vibrational and rotational temperatures, which are found to be 3200 K and 340 K respectively. As a measure of pollution control potential of the constructed pulsed corona system, NO oxidation efficiency was investigated and compared with results presented in literature. It was shown that pulsed corona systems with significantly longer pulse durations are competitive with several times shorter pulse duration systems, which implies that chemical efficiency of secondary streamers is comparable with efficiency of primary streamers.     

Spatial and temporal variability of stable isotopes and biological parameters for the Danube River in Serbia

This paper presents the results of hydrological, physicochemical, biological, and isotopic investigations of the Danube River along the stretch through Serbian territory conducted during four campaigns in September and November 2007, September 2008 and April 2009. The stable isotope values exhibited significant changes both in the Danube (?10.7 to?9.5‰ for δ¹⁸O and?73.7 to?67.1 ‰ for δ²H) and in its tributaries (?9.1 to?8.5‰ for δ¹⁸O and?69.4 to?59.4‰ for δ²H) depending on the time of survey, which could be partly attributed to the influences of seasonal effects. Results emphasise the dominant role of tributaries inflows from aquifers along the Danube. The very narrow range of δ¹³C_POC (from?28.9 to?27.4 ‰) was associated with relatively high C/N ratios (C/N>9), and together with δ¹⁵N_TPN values, the date suggested that, in early spring, a major fraction of particulate organic matter was derived from allochthonous matter. An orthogonal varimax rotation of the principal components analysis identified four latent factors (‘mineral related’, ‘biological’, ‘hardness’, and ‘soil inlets’) which are responsible for the data structure covering 79% of the observed variations among the variables studied. A reliable grouping of samples with respect to the season was found.     

Recent advances in the application of high performance liquid chromatography in the analysis of polyphenols in wine and propolis

Wine and propolis are among the richest sources of polyphenolics. Flavonoids and phenolic acids are main pharmacological components whose benefits have been shown in numerous reports. This paper presents a review of the latest applications of HPLC for determination of polyphenolics in propolis and wine. Special attention is focused on sample preparation, chromatographic conditions, and detection of polyphenolics. HPLC will undoubtedly be the most used method that is readily available for analysis, as well as for isolation of polyphenolics in complex mixtures.     

Effects of St John's wort (Hypericum perforatum L.) extracts on epileptogenesis

The purpose of this study was to investigate the effects of treatment with water, n-butanol and ether extracts of Hypercom perforatum L. on epileptogenesis in rabbits. Animals from the control group received solvent-ethanol, and the kindling model of epilepsy was used. Epileptic focus was induced in Chinchilla rabbits by stimulation of the hippocampus. The following parameters were determined: the minimum current strength necessary to induce after-discharge (AD) - discharges appearing after cessation of stimulation; AD duration; the number of stimulations necessary to induce spontaneous kindling; and the latency time for the development of full kindling. The results obtained indicate that epileptogenesis is influenced by Hypericum perforatum L. extract treatment. Animals treated with an ether extract of Hypericum perforatum L. required significantly weaker minimum current strengths for the development of epileptogenic focus, and displayed longer AD times, while the number of electro-stimulations necessary for full kindling was less. In contrast, animals treated with water and n-butanol extracts required increased electro-stimulations for the development of epileptic discharge, and displayed shortened AD durations versus controls.     

Quantitative estimation of ultrasound beam intensities using infrared thermography-Experimental validation

Infrared (IR) thermography is a technique that has the potential to rapidly and noninvasively determine the intensity fields of ultrasound transducers. In the work described here, IR temperature measurements were made in a tissue phantom sonicated with a high-intensity focused ultrasound (HIFU) transducer, and the intensity fields were determined using a previously published mathematical formulation relating intensity to temperature rise at a tissue/air interface. Intensity fields determined from the IR technique were compared with those derived from hydrophone measurements. Focal intensities and beam widths determined via the IR approach agreed with values derived from hydrophone measurements to within a relative difference of less than 10%, for a transducer with a gain of 30, and about 13% for a transducer with a gain of 60. At axial locations roughly 1 cm in front (pre-focal) and behind (post-focal) the focus, the agreement with hydrophones for the lower-gain transducer remained comparable to that in the focal plane. For the higher-gain transducer, the agreement with hydrophones at the pre-focal and post-focal locations was around 40%.     

High-resolution mass spectrometry and hydrogen/deuterium exchange study of mitorubrin azaphilones and nitrogenized analogues

Azaphilones represent numerous groups of wild fungal secondary metabolites that exhibit exceptional tendency to bind to nitrogen atoms in various molecules, especially those containing the amine group. Nitrogenized analogues of mitorubrin azaphilones, natural secondary metabolites of Hypoxylon fragiforme fungus, have been detected in the fungal methanol extract in very low concentrations. Positive electrospray ionization interfaced with high-resolution mass spectrometry was applied for confirmation of the elemental composition of protonated species. Collision-induced dissociation (CID) experiments have been performed, and fragmentation mechanisms have been proposed. Additional information regarding both secondary metabolite analogue families has been reached by application of gas-phase proton/deuterium (H/D) exchanges performed in the collision cell of a triple quadrupole mass spectrometer. An incomplete H/D exchange with one proton less than expected was observed for both protonated mitorubrin azaphilones and their nitrogenized analogues. By means of the density functional theory, an appropriate explanation of this behavior was provided, and it revealed some information concerning gas-phase H/D exchange mechanism and protonation sites. Copyright ? 2012 John Wiley & Sons, Ltd.