Cyclic AMP Receptor Protein from Yeast Mitochondria

We have identified and characterized a cyclic AMP receptor protein in mitochondria of the yeast Saccharomyces cerevisiae. The binding is specific for cyclic nucleotides, particularly for cyclic AMP which is bound with high affinity (Kd of 10(-9) M) at 1 to 5 pmol/mg of mitochondrial protein. The mitochondrial cyclic AMP receptor is synthesized on cytoplasmic ribosomes and has an apparent molecular weight of 45,000 as determined by photoaffinity labeling. It is localized in the inner mitochondrial membrane and faces the intermembrane space. Cross-contamination of mitochondrial inner membranes by plasma membranes or soluble cytoplasmic proteins is excluded

The arabidopsis COX11 homolog is essential for cytochrome c oxidase activity

Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or over expression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor.Fil: Radin, Ivan. Technische Universität Dresden; AlemaniaFil: Mansilla, Natanael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Rödel, Gerhard. Technische Universität Dresden; AlemaniaFil: Steinebrunner, Iris. Technische Universität Dresden; Alemani

Die Zellatmung: ein effizienter biologischer Prozess nicht ohne Risiken

The energy supplies of modern society seem to be necessarily associated with risks. In this paper, we address the question as to whether the efficient utilisation of energy in biological systems is also coupled to hazards. Most organisms oxidise highly reduced substrates with molecular oxygen in order to gain energy. In eukaryotes, this process takes place at the inner membrane of specialised organelles, the mitochondria. Interestingly, about 1% of the consumed oxygen molecules are reduced not to water, but to ROS (reactive oxygen species), which are deleterious to many macromolecules, including mitochondrial DNA. As a result, mitochondrial DNA mutations accumulate, in turn affecting the energy supply and inducing degeneration and ageing.Nach dem heutigen Stand der Technik scheint eine ausreichende Energieversorgung der Gesellschaft stets mit Risiken verbunden zu sein. Wir gehen in dem Artikel der Frage nach, ob auch biologische Systeme bei der Nutzung von Energie Risiken in Kauf nehmen. Zur effizienten Energiegewinnung nutzen Organismen meist Sauerstoff zur Oxidation energiereicher Substrate. In Eukaryonten erfolgt die Energiegewinnung vor allem an der inneren Membran von Mitochondrien. Etwa 1 % des verbrauchten Sauerstoffs wird dabei nicht zu Wasser, sondern zu ROS (reactive oxygen species, reaktive Sauerstoffspezies) reduziert, die unter anderem die mitochondriale DNA schädigen und Mutationen hervorrufen. Diese akkumulieren auf Dauer und führen zu einer Störung der Energiegewinnung, in deren Folge Degenerations- und Alterungsphänomene auftreten

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

BACKGROUND: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. RESULTS: Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter. CONCLUSION: The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism

Calcium Alginate Gels as Stem Cell Matrix - Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300, 000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs

Calcium Alginate Gels as Stem Cell Matrix - Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300, 000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs

A large aperture reflective wave-plate for high-intensity short-pulse laser experiments

We report on a reflective wave-plate system utilizing phase-shifting mirrors (PSM) for a continuous variation of elliptical polarization without changing the beam position and direction. The scalability of multilayer optics to large apertures and the suitability for high-intensity broad-bandwidth laser beams make reflective wave-plates an ideal tool for experiments on relativistic laser-plasma interaction. Our measurements confirm the preservation of the pulse duration and spectrum when a 30-fs Ti:Sapphire laser beam passes the system

Characterization of encapsulated graphene layers using extreme ultraviolet coherence tomography

Many applications of two-dimensional materials such as graphene require the encapsulation in bulk material. While a variety of methods exist for the structural and functional characterization of uncovered 2D materials, there is a need for methods that image encapsulated 2D materials as well as the surrounding matter. In this work, we use extreme ultraviolet coherence tomography to image graphene flakes buried beneath 200 nm of silicon. We show that we can identify mono-, bi-, and trilayers of graphene and quantify the thickness of the silicon bulk on top by measuring the depth-resolved reflectivity. Furthermore, we estimate the quality of the graphene interface by incorporating a model that includes the interface roughness. These results are verified by atomic force microscopy and prove that extreme ultraviolet coherence tomography is a suitable tool for imaging 2D materials embedded in bulk materials

A high resolution extreme ultraviolet spectrometer system optimized for harmonic spectroscopy and XUV beam analysis

We present a modular extreme ultraviolet (XUV) spectrometer system optimized for a broad spectral range of 12-41 nm (30-99 eV) with a high spectral resolution of λ/Δλ 784 ± 89. The spectrometer system has several operation modes for (1) XUV beam inspection, (2) angular spectral analysis, and (3) imaging spectroscopy. These options allow for a versatile use in high harmonic spectroscopy and XUV beam analysis. The high performance of the spectrometer is demonstrated using a novel cross-sectional imaging method called XUV coherence tomography. © 2019 Author(s)

Absolute EUV reflectivity measurements using a broadband high-harmonic source and an in situ single exposure reference scheme

We present a tabletop setup for extreme ultraviolet (EUV) reflection spectroscopy in the spectral range from 40 to 100 eV by using high-harmonic radiation. The simultaneous measurements of reference and sample spectra with high energy resolution provide precise and robust absolute reflectivity measurements, even when operating with spectrally fluctuating EUV sources. The stability and sensitivity of EUV reflectivity measurements are crucial factors for many applications in attosecond science, EUV spectroscopy, and nano-scale tomography. We show that the accuracy and stability of our in situ referencing scheme are almost one order of magnitude better in comparison to subsequent reference measurements. We demonstrate the performance of the setup by reflective near-edge x-ray absorption fine structure measurements of the aluminum L2/3 absorption edge in α-Al2O3 and compare the results to synchrotron measurements