Hopping in the electron-transfer photocycle of the 1:1 complex of Zn-cytochrome c peroxidase with cytochrome c

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants: X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which "short-circuits" the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.     

Absence of crystalline ordering in two dimensions

We give conditions on the potential of a classical particle system, which imply absence of crystalline ordering in two dimensions. We thereby correct and extent some results in a previous paper.J. Fröhlich: Comment on Absence of Crystalline Ordering in two Dimensions     

Determination of fluazifop-butyl and fluazifop with the use of disposable solid phase extraction columns for selective clean-up and concentration of Soxhlet soil extracts

Summary An analytical procedure based on the solid phase extraction technology has been developed for the clean-up and concentration of Soxhlet soil extracts containing fluazifop-butyl and fluazifop by the use of a phenyl phase cartridge. No liquid-liquid partition has been used; thus the consumption of organic solvents was limited and the use of chlorinated solvents could be avoided. Quantification has been performed by ion-pair HPLC. Despite the large difference in polarity the recoveries of both the compounds from spiked soil samples between 0.1 and 1 g/g was higher than 90%. The solid-phase adsorption technology resulted in a very effective methodology of clean-up in the case of the polar compound fluazifop, for which a second disposable column with a cyanopropyl phase has been used, and was fairly satisfactory for fluazifop-butyl. The detection limits were less than 0.04 g/g and 0.10 g/g, respectively for fluazifop and fluazifop-butyl.     

Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones

Quorum sensing (QS) is a communication mechanism between bacteria using diffusible chemical signaling molecules, which are called autoinducers (AI). By detecting the concentration of quorum sensing molecules through binding to a specific receptor protein, bacteria regulate their gene expressions when the concentration of autoinducers and thus the cell density reaches a threshold level. Many Gram-negative bacteria use acylated homoserine lactones (HSLs) as autoinducers. Because of the broad biological functions of HSLs, interest in detection and analysis of HSLs is increasing with a view to their medical, biotechnological, and agricultural applications. In this study, an anti-HSL antibody-based immunochemical detection method has been developed. Four structurally distinct HSL haptens, named HSL1, HSL2, HSL3, and HSL4, have been designed for antibody and assay development. New rat anti-HSL monoclonal antibodies (mAbs) have been produced in-house and characterized with enzyme-linked immunosorbent assays (ELISA), both in the coating antigen and in the enzyme tracer format. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5E12, HSL4-5H3, and HSL4-6D3) will be presented in this paper. We demonstrate that the anti-HSL mAbs have distinguished sensitivity and selectivity toward HSLs depending upon their chemical structures. The optimized assays are capable of detecting HSLs in the microgram per liter (low micromolar to nanomolar) range. The best IC₅₀ (test midpoint) was 134 ± 30 μg L⁻¹ (n = 54) for N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) using mAb HSL1/2-2C10 and HSL1–HRP in the enzyme tracer format. In the coating antigen format, the most selective mAb for N-octanoyl-l-homoserine lactone (C8-HSL) was mAb HSL4-4C9. Additionally, anti-HSL mAbs showed higher sensitivity against hydrolyzed HSLs, namely homoserines. These compounds might also occur under certain biological conditions. This study marks the beginning of new ways for quick and cost-effective HSL detection, requiring small sample amounts (less than 1 mL) and little to no sample preparation.     

On axially symmetric solutions of Einstein's field equations in matter and an example of static gravitational shielding

A model system, consisting of a thin spherical shell with radiusR and massM and a point massm at a distances>R from the center of the sphere, held fixed by an appropriate strut, is solved to ordermM. The stresses in the shell are not of the canonical Weyl type, and it is argued that the same is true for more realistic situations, e.g., rotating matter. Owing to the nonlinearity of Einstein's field equations, the field of the point mass is shielded from the interior of the shell by a factor lying between 1–3M/R and 1–2M/R, and the field outside the shell explicitly depends onR.     

1,4,6,9‐Tetraoxa‐5λ4‐selena‐spiro[4.4]nonane – A Combined Theoretical and Experimental Study

The symmetric spiro‐selenurane derived from ethylene glycol, 1,4,6,9‐tetraoxa‐5λ⁴‐selena‐spiro[4.4]nonane, was prepared from selenium tetrachloride and ethylene glycol and its molecular structure was determined by single crystal X‐ray diffraction. NBO analyses for the title compound and a related compound were conducted to assess the role of the stereochemical active lone pair on the selenium atom on the structure.     

Equilibrium shapes of crystals attached to walls

We discuss equilibrium shapes of crystals attached to walls. Optimal shapes for different configurations of walls are found and the minimality of the overall surface tension is proven with the help of a simple geometrical argument based on the isoperimetric inequality and monotonicity. Stability results in the form of Bonnesen inequalities are obtained in the two-dimensional case.     

Subsurface Microscopical visualization of brain tissue in vivo: Present, problems and prospects

The location of the brain within the cranium has prevented in vivo studies with microscopical resolution for a long time. Cranial window techniques provide microscopical access to the brain surface, but subsurface structures cannot be visualized with conventional microscopy. Confocal microscopy with its increased depth of penetration and optical sectioning capabilities is ideally suited for the investigation of thick biological specimens. Due to the scanning process, however, temporal resolution is limited, a significant disadvantage in the in vivo setting. In this article we demonstrate that confocal laser scanning microscopy can be utilized to study brain cortex microvascular morphology, capillary hemodynamics, leukocyte behaviour and intracellular ion concentrations in anesthetized rats through a closed cranial window. Three different confocal microscopes are compared: a Biorad MRC-600 with multiline Kr/Ar-Laser (488/568/647 nm), a Noran Odyssey acousto-optic scanning microscope with multiline Ar-Laser (458/488/514/529 nm) and a Biorad Viewscan DVC-250 slit scanning microscope with Ar-Laser (488/514 nm). With all microscopes a Zeiss × 40 water immersion objective, n.a. 0.75 is used. A Laser-Doppler flowmeter continuously measures regional cerebral blood flow in the area of microscopical investigation. As fluorescent dyes we used: fluorescein sodium as blood plasma marker (given I.V.); rhodamine 6G to label leukocytes (given I.V.); and the AM-esters of BCECF (pH-sensitive), Fluo-3 and Calcium-Green (both calcium-sensitive) as intracellular ion-concentration markers (loaded via superfusion over the cranial window). With this setup, we are able to study the flow dynamics in the capillary network of the cortex (erythrocyte flow velocities and flux rates), the behaviour of leukocytes in capillaries and postcapillary veins (plugging of capillaries, adhesion to the endothelium, extravasation into the parenchyma), and intracellular changes of [H⁺] and [Ca²⁺] under physiological and pathophysiological conditions (cerebral ischemia and meningitis). The comparison between the conventional CLSM (Biorad 600) and the real time CLSMs revealed that the increase in temporal resolution afforded by the real time instruments is offset by a reduction in spatial resolution and, most importantly, in the signal to noise ratio, resulting in a lower depth of penetration into the tissue and necessitating frame averaging.