Transport of glutathione across the endoplasmic reticulum membrane

γ-L-glutamyl-L-cysteinylglycine known as glutathione is tripeptide synthesised in the cytoplasm. Glutathione plays important role in several cellular processes and has many functions, for example detoxification of xenobiotics, oxidative protein folding, protection against reactive oxygen species, modulation of cell proliferation and apoptosis. Glutathione is present in almost all organelles within the cell, however, it is still not known how glutathione is transported from the cytoplasm to other cellular compartments. Here we investigated the transport of glutathione into the ER where glutathione plays an important role in oxidative protein folding. Two assays to monitor glutathione transport across the ER membrane were developer and an attempt to identify the putative glutathione transporter was made. Both assays rely on selective permeability of biological membranes and use microsomes to mimic the ER environment. Microsomes were prepared from HT1080 cells expressing either redox sensitive green fluorescent protein (roGFP-iE) or glutathione S-transferase P1 (GSTP1-1A) inside the ER. By measuring the change in fluorescence of roGFP-iE it was possible to measure the rate at which glutathione is transported inside microsomes. GSTP1-1A is able to conjugate glutathione to various substrates and form a stable product, by measuring the increase in glutathione conjugates it was possible to estimate the transport of glutathione inside microsomes. Using both roGFP-iE and GSTP1-1A based assays we were able to measure glutathione transport into microsomes, both assays provide slightly different information about glutathione transport and both have different limitations. In order to identify the glutathione transporter, we used GSH as an affinity ligand and isolated all ER membrane proteins interacting with glutathione. The first approach using glutathione Sepharose beads allowed to isolate several proteins, however, all of them belonged to GST family. A second approach relied on isolating the transporter using glutathione attached to Mts-Aft-Biotin a photo activated crosslinker. Mts-Aft-Biotin approach did not result in isolation of any proteins binding specifically to GSH, this approach requires more work and needs to be improved. We showed that it is possible to isolate GSH binding proteins using GSH as affinity ligand, using this strategy it might be possible to isolate GSH transporter in the future. Both assays presented in this work are the first assays specific for the transport of glutathione across the ER membrane. In the future these assays can be used to investigate the transport of glutathione even further and contribute to the understanding of redox homeostasis in the ER

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane 1 protein to reduce ER-localised proteins

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis ensuring correct protein folding and facilitating the degradation of misfolded ER proteins

Detection Limits of DLS and UV-Vis Spectroscopy in Characterization of Polydisperse Nanoparticles Colloids

Dynamic light scattering is a method that depends on the interaction of light with particles. This method can be used for measurements of narrow particle size distributions especially in the range of 2–500 nm. Sample polydispersity can distort the results, and we could not see the real populations of particles because big particles presented in the sample can screen smaller ones. Although the theory and mathematical basics of DLS technique are already well known, little has been done to determine its limits experimentally. The size and size distribution of artificially prepared polydisperse silver nanoparticles (NPs) colloids were studied using dynamic light scattering (DLS) and ultraviolet-visible (UV-Vis) spectroscopy. Polydisperse colloids were prepared based on the mixture of chemically synthesized monodisperse colloids well characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), DLS, and UV-Vis spectroscopy. Analysis of the DLS results obtained for polydisperse colloids reveals that several percent of the volume content of bigger NPs could screen completely the presence of smaller ones. The presented results could be extremely important from nanoparticles metrology point of view and should help to understand experimental data especially for the one who works with DLS and/or UV-Vis only

Działalność pozawystawiennicza muzeów na przykładzie projektów społecznych wybranych muzeów krakowskich

Tematem pracy są projekty społeczne muzeów w Krakowie i rola muzeów we współczesnym świecie. W pracy omówione zostały najważniejsze pojęcia związane z muzealnictwem. Przedstawiona jest historia muzeów oraz ich zadania, a także – co szczególnie istotne z perspektywy niniejszej pracy - działalność pozawystawiennicza. Szczegółowej analizie poddano aktywność muzeów w realizacji projektów społecznych. Zasadniczą częścią pracy jest prezentacja najciekawszych projektów społecznych trzech muzeów krakowskich: Muzeum Narodowego, Muzeum Etnograficznego im. Seweryna Udzieli oraz Muzeum Sztuki Współczesnej MOCAK. Na koniec przedstawiona jest działalność innego rodzaju instytucji kultury-domu kultury i jego oferty. Opisany jest projekt społeczny Domu Kultury im. C.K. Norwida w Krakowie. Głównym celem pracy było przedstawienie nowej dziedziny działań muzeów.The theme of this thesis deals with social projects of museums in Krakow and their role in modern life. In my work are discussed the key concepts connected with museology, the history of museums and their activities outside the exhibition area like also main functions which are undertaken by museums nowadays. Krakow city, its conditions and cultural policy, was taken as subject of the researches. An essential part of my work is presentation of the most interested social projects of three museums in Krakow: National Museum, Ethnographical Museum named Seweryn Udziela and Museum Of Contemporary Art MOCAK. At the end of my work is introducing another kind of cultural institution - Community Center and its offer. I analyzed social project represented by C.K. Norwid Community Center in Krakow. The main objective of this work was presentation of new courses and activities of museums

Effect of surfactant on electrochemical properties of supercapacitor

Napięcie powierzchniowe cieczy ma istotny wpływ na transport masy i energii na granicy międzyfazowej. W superkondensatorze, gdzie ładunek gromadzony jest dzięki istnieniu podwójnej warstwy elektrycznej na granicy faz pomiędzy elektrodą a elektrolitem, napięcie powierzchniowe elektrolitu wpływa na zwilżalność i powinowactwo z materiałem węglowej elektrody. Poprawa zwilżalności elektrody przez elektrolit zwiększa dostępność porowatych powierzchni elektrod węglowych dla jonów elektrolitu. Dzięki temu możliwie jest zgromadzenie większej liczby ładunków oraz obniżenie łącznej, wewnętrznej oporności urządzenia. W pracy przedstawiono wpływ zmniejszenia napięcia powierzchniowego – poprawę zwilżalności materiału węglowego elektrolitem na właściwości elektrochemiczne superkondensatora. Zmiany napięcia powierzchniowego realizowano dodając do elektrolitu surfaktanty. Surfakanty dodawano poniżej krytycznego stężenia micelarnego, aby uniknąć tendencji do tworzenia agregatów, które mogą blokować pory węglowej elektrody. Właściwości elektrochemiczne celki kondensatora sprawdzono metodami woltametrii cyklicznej, galwanostatycznego ładowania i rozładowania oraz spektroskopii impedancyjnej.The surface tension of liquids has a significant impact on the transport of mass and energy at the interface. In case of supercapacitor, where the charge is collected by the existence of electrical double layer at the interface between electrode and electrolyte, surface tension of an electrolyte reflects the wettability and affinity for the electrode material. The improvement of the wettability of electrode in the electrolyte increases the access of electrolyte to the surface of porous carbon electrode and accordingly better charge accumulation is achieved. It also results in a lower internal resistance of cell. In this work, impact of decreasing surface tension and increasing the electrolyte wetting properties on the electrochemical properties of supercapacitor were investigated. Improvement of these properties has been achieved through the inclusion of surfactant to electrolyte. Surfactants were added under critical micelle concentrations (CMC) to avoid surfactant tendency to form aggregates, that can block the pore in carbon electrode. Properties of electrochemical capacitor cell by cyclic voltammetry methods, galvanostatic charge and discharge and impedance spectroscopy were measured

On Temperature Dependence of Domain Structure in Cobalt

The type-I magnetic contrast in a scanning electron microscope is used to study the domain behaviour on the basal planes of cobalt monocrystals of different thicknesses during a heating cycle. Digital image processing is applied to the original scanning electron microscope images for their restoration, enhancement and analysis. The main reasons for the application of digital image processing are: low level of type-I magnetic contrast, particularly used at the higher temperatures, and the complex character of the magnetic domain structure. The changes in both domain structures and type-I magnetic contrast are due to the strong temperature dependence of magnetocrystalline anisotropy energy for the hcp phase of cobalt, which implies the rotation of magnetic easy axis from the c-axis to the basal plane. The temperature of the magnetic phase transition between an open-flux and a closed-flux domain configuration was found to be dependent on the specimen thickness. The changes in domain structure during the heating cycle were reversible under the condition that the specimen was not carried through the hcp-to-fcc phase transition. Otherwise, they were partially or completely irreversible and were caused by crystal imperfections originating from the transition. The paper shows the great advantage of using digital image processing system for data restoration, enhancement and analysis