Reactions of Nucleosides with 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU). Syntheses of N(3)-Methylene-Bridged Bis-uridines and Secouridine-Dinucleoside Analogs

Diasteroisomeric secouridine derivatives, appropriately protected and activated, served as starting compounds in the reactions with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in various solvents (CH₂Cl₂, MeCN, or dimethylformamide (DMF)). Reactions with DBU/CH₂Cl₂ gave N(3)-methylene-bridged bis-secouridines and bis-uridines (Scheme 3), while the reactions with DBU in non-alkylating solvents resulted in formation of secdinucleosides as the result of intermolecular ‘dimerizations’ (Scheme 2).     

Observation of parity violating effects in the polarized thermal neutron total and radiative capture cross sections of 117Sn and 139La

Parity violating effects in the transmission and radiative capture of longitudinally polarized thermal neutrons have been observed for ¹¹⁷Sn and ¹³⁹La targets. Helicity dependent asymmetries a = 10⁶ (σ⁺-? σ^?)/(σ⁺ + σ^?) were found in the total cross section of ¹¹⁷Sn (a = 6.2 ± 0.7) and ${}^{139}\text{La}\text{(a = 9.0 ± 1.4)}$, and also in the (n, γ) cross section of ¹¹⁷Sn (a = 22.6 ± 1.9) and of ¹³⁹La (a = 16.1 ± 2.0).     

Constructibility Classes for Triangle Location Problems

Straightedge-and-compass construction problems are well known for different reasons. One of them is the difficulty to prove that a problem is not constructible: it took about two millennia to prove that it is not possible in general to cut an angle into three equal parts by using only straightedge and compass. Today, such proofs rely on algebraic tools difficult to apprehend by high school student. On the other hand, the technique of problem reduction is often used in theory of computation to prove other kinds of impossibility. In this paper, we adapt the notion of reduction to geometric constructions in order to have geometric proofs for unconstructibility based on a set of problems known to be unconstructible. Geometric reductions can also be used with constructible problems: in this case, besides having constructibility, the reduction also yields a construction. To make the things concrete, we focus this study to a corpus of triangle location problems proposed by William Wernick in the eighties.     

Kinetics of inhibition of peroxidase activity of myeloperoxidase by quercetin

The inhibition of myeloperoxidase (MPO), isolated from human neutrophils, by quercetin was investigated by following peroxidase activity of the enzyme using o-dianisidine as the substrate. The inhibition parameters (IC₅₀) were obtained by graphical analysis of the inhibition curves. A reaction mechanism, which involved the enzyme inhibition by quercetin and H₂O₂ in excess, was proposed. The rate and equilibrium constants for the proposed reaction path were calculated from experimental data. Kinetic analysis in noninhibiting H₂O₂ concentration range in the absence and the presence of quercetin revealed that the reaction mechanism underwent Michaelis–Menten kinetics. K and V values indicated that quercetin was a mixed inhibitor of MPO activity. The initial reaction rates were recalculated using the obtained results. Calculated curves fitted the experimental results within the range of experimental error. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 384–394, 2008     

Electroformation in a flow chamber with solution exchange as a means of preparation of flaccid giant vesicles

A recently described technique [Estes and Mayer, Biochim. Biophys. Acta 1712 (2005) 152-160] for the preparation of giant unilamellar vesicles (GUVs) in solutions with high ionic strength is examined. By observing a series of osmotic swellings followed by vesicle bursts upon a micropipette transfer of a single POPC GUV from a sucrose solution into an iso-osmolar glycerol solution, a value for the permeability of POPC membrane for glycerol, P=(2.09+/-0.82) x 10(-8)m/s, has been obtained. Based on this result, an alternative mechanism is proposed for the observed exchange of vesicle interior. With modifications, the method of Estes and Mayer is then applied to preparation of flaccid GUVs.     

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.