Digital Variance Angiography in Lower-Limb Angiography with Metal Implants.

PURPOSE: The presence of metal implants may reduce angiographic image quality due to automated beam adjustments. Digital variance angiography (DVA) is reported to be superior to digital subtraction angiography (DSA) with increased contrast-to-noise ratio (CNR) and better image quality. The aim of the study was to evaluate whether DVA could counterbalance the image quality impairment of lower-limb angiographies with metal implants. MATERIALS AND METHODS: From November 2019 to January 2020, 85 raw lower-limb iodine contrast angiograms of 12 patients with metal implants were processed retrospectively with DVA analyses. For objective comparison, CNR of DSA and DVA images was calculated and the ratio CNRDVA/CNRDSA was determined. Visual image quality was evaluated in a paired comparison and by a five-grade Likert scale by three experienced radiologists. RESULTS: The CNR was calculated and compared in 1252 regions of interest in 37 image pairs containing metal implants. The median ratio of CNRDVA/CNRDSA was 1.84 with an interquartile range of 1.35-2.32. Paired comparison resulted in 84.5% in favour of DVA with an interrater agreement of 83.2% (Fleiss κ 0.454, p < 0.001). The overall image quality scores for DSA and DVA were 3.64 ± 0.08 and 4.43 ± 0.06, respectively (p < 0.001, Wilcoxon signed-rank test) with consistently higher individual ratings for DVA. CONCLUSION: Our small-sample pilot study shows that DVA provides significantly improved image quality in lower-limb angiography with metal implants, compared to DSA imaging. The improved CNR suggest that this approach could reduce radiation exposure for lower-limb angiography with metal implants. LEVEL OF EVIDENCE: Level 4, case studies.

Aromatic substrate specificity of horseradish peroxidase C studied by a combined fluorescence line narrowing/energy minimization approach: the effect of localized side-chain reorganization.

Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on high-resolution fluorescence line-narrowed spectra which show that the effects of substrate binding on the electronic transitions are similar for both substrates and present data on the normal vibrational modes that are active in the vibronic spectra. Analysis of the vibrational frequencies shows that the Mg(II) ion is 5-coordinate in all cases, thus ruling out a solvent water as sixth ligand. The frequency shifts observed as a result of substrate binding are also indicative of a more rigid prosthetic group upon substrate binding. We present models for MgMP-HRP and its complexes with both substrates and compare the resulting structures on the basis of a modeling approach combining energy minimization to finite difference Poisson--Boltzmann calculations which partitions the various relative protein contributions to substrate binding. We show that the electrostatic potential of the prosthetic group is modified by the binding event. Analysis of the unbound and bound energy-minimized structures shows that the enzyme modulates substrate binding by subtle charge reorganization in the vicinity of the catalytic site and that this rearrangement is not attributable to significant secondary structure conformational changes but to side-chain reorganization.

Site specific labeling of Rhodobacter sphaeroides reaction centers with dye probes for surface pH measurements.

Covalently bound pH sensitive dyes are an important tool for characterizing the proteolytic reactions of protein complexes that play key roles in biological energy transduction. Here we demonstrate the feasibility of this method for photosynthetic reaction centers (RCs) for the first time, by the highly selective attachment of two thiol reactive derivatives of fluorescein to the two H subunit cysteines of the photosynthetic RC from Rhodobacter sphaeroides R-26 The pK(a) shifts of the dyes upon binding to the protein and in response to high salt were measured, and interpreted based on the structure of the RC. 2-[(5-fluoresceinyl)aminocarbonyl]ethyl-methanethiosulfonate was attached to Cys H156 and fluorescein-5-maleimide to Cys H234. By following the absorption changes of bound fluorescein (500 nm), and those of the hydrophilic pH indicator 8-hydroxypyrene-1,3,6-tris-sulfonic acid (468 nm), the surface and bulk pH were monitored separately with less than 5% crosstalk. Flash-induced proton uptake and external calibrations by mixing with aliquots of acid were measured in different redox states of the RCs. The results indicate that the charge in the quinone acceptor complex after flash activation (primary quinone acceptor (Q(A))- or secondary quinone acceptor (Q(B))-) has no effect on the surface pH and potential in the vicinity of these two attachment sites, between pH 6.5 and 9. Application of the method to other surface locations is discussed.

Coupling of cytochrome and quinone turnovers in the photocycle of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides.

A minimal kinetic model of the photocycle, including both quinone (Q-6) reduction at the secondary quinone-binding site and (mammalian) cytochrome c oxidation at the cytochrome docking site of isolated reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides, was elaborated and tested by cytochrome photooxidation under strong continuous illumination. The typical rate of photochemical excitation by a laser diode at 810 nm was 2.200 s-1, and the rates of stationary turnover of the reaction center (one-half of that of cytochrome photooxidation) were 600 +/- 70 s-1 at pH 6 and 400 +/- 50 s-1 at pH 8. The rate of turnover showed strong pH dependence, indicating the contribution of different rate-limiting processes. The kinetic limitation of the photocycle was attributed to the turnover of the cytochrome c binding site (pH < 6), light intensity and quinone/quinol exchange (6 < pH < 8), and proton-coupled second electron transfer in the quinone acceptor complex (pH > 8). The analysis of the double-reciprocal plot of the rate of turnover versus light intensity has proved useful in determining the light-independent (maximum) turnover rate of the reaction center (445 +/- 50 s-1 at pH 7.8).

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer.

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.

Trapping magnetically oriented chloroplast thylakoid membranes in gels for electric measurements.

Electrochromic absorbance change and light gradient photovoltage measurements were carried out in chloroplast thylakoid membranes embedded in different compositions of gels. The goal was to find a system suitable for determining the dependence of the amplitude of the anomalous light gradient photovoltage signal, with opposite sign with respect to the ordinary signal, on the alignment of membranes. We found that polyacrylamide gel drastically increased the permeability of membranes which rendered electric measurements impossible in this gel. Agarose gel was successfully used in electric measurements. We will show that the light gradient photovoltage induced by a 580 nm laser flash depends strongly on the alignment of membranes with respect to the direction of the excitation beam. With face-aligned membranes the amplitude of the anomalous (negative) signal was drastically diminished, and the ordinary (positive) signal became clearly discernible. The observed differences in the decay of the two signals are tentatively explained by differences in the rates of ionic equilibration between different subcompartments of the inner aqueous phase of the thylakoid membrane system.

The immunobiology of murine interleukin-1 alpha encoded by recombinant vaccinia virus.

Recombinant vaccinia viruses were constructed which encoded murine interleukin-1 alpha (IL-1 alpha) (VV-IL1). One virus also encoded the hemagglutinin (HA) gene of influenza virus (VV-HA-IL1). Mice were infected with these viruses and the effects of co-expressed IL-1 on various immune parameters were assessed. The growth of VV-IL1 in vivo was less than that of the control virus, and this was reflected in the reduced virus-induced cell-mediated immune responses. However, specific antibody responses generated after challenge with vaccinia or influenza viruses were significantly higher when VV-HA-IL1 was used to prime mice, compared to the control virus (VV-HA-TK). This study demonstrates that co-expressed cytokines may be useful for selective alteration of immune reactivity.

Intratubular germ cell tumours of pig testicles.

In the course for the histopathological examination of testicles of 30 castrated boars the authors have observed in 4 animals the intratubular growth of atypical germ cells. On the basis of the histological picture this change is equivalent to the human infertile testicles, observed as carcinoma in situ, resp. intratubular germ cell tumour. The described phenomenon occurred in atrophic testicles in 3 of the 4 old animals. The described change in the domestic pig, not known so far, raises the possibility of development of solid testis tumours in this species, as well.