Sidedness of Biosynthesis of Glycosylphosphatidylinositol Anchors in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Many surface membrane glycoproteins of eucaryotes are attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthesis of these anchors proceeds through two stages. First, the synthesis of the protein and of a free glycosylphosphatidylinositol (GPI) is achieved separately. In a second step, the protein is hooked onto the preformed free GPI whereby a provisional C-terminal hydrophobic peptide is removed. The GPI-anchored protein is subsequently transported to the cell surface by way of vesicular traffic. It is presumed that the attachment of the preformed free GPI's to proteins occurs on the luminal surface of the endoplasmic reticulum (ER). The stepwise addition of sugars by glycosyltransferases onto phosphatidylinositol to form a free GPY is equally presumed to occur in the ER, but it is unclear whether these reactions take place at the cytosolic or the luminal side of the membrane. Here we tried to get some information on the membrane orientation of free GPYs in Saccharomyces cerevisiae surmising that their orientation might tell us something about the probable location of the biosynthetic process. When using trinitrobenzenesulfonic acid as a probe, we find that 75% of the free GPIs in intact ER-derived microsomes get derivatized, whereas 100% get derivatized in detergent-permeabihzed microsomes. This finding is compatible with the idea that in yeast lipid anchors are built up at the cytosolic surface

Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF-beta 1 activation and reduces regulatory ID gene expression

Tissue inhibitor of metalloproteinase 1 (TIMP-1) controls matrix metalloproteinase activity through 1:1 stoichiometric binding. Human TIMP-1 fused to a glycosylphosphatidylinositol (GPI) anchor (TIMP-1-GPI) shifts the activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1-GPI treated renal cell carcinoma cells show increased apoptosis and reduced proliferation. Transcriptomic profiling and regulatory pathway mapping were used to identify the potential mechanisms driving these effects. Significant changes in the DNA binding inhibitors, TGF-beta 1/SMAD and BMP pathways resulted from TIMP-1-GPI treatment. These events were linked to reduced TGF-beta 1 signaling mediated by inhibition of proteolytic processing of latent TGF-beta 1 by TIMP-1-GPI

Removal of direct blue 129 from aqueous medium using surfactant-modified zeolite: a neural network modeling

Background: Conserving water for human survival and providing future security are important issues that need to be addressed. Methods: In this study, a zeolite modified with hexadecyl trimethyl ammonium bromide (HDTMA-Br), a cationic surfactant, and its application in removing direct blue 129 (DB129) was examined. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) were used to characterize both modified and unmodified zeolites. The effects of operational parameters such as the amount of adsorbent, initial dye concentration and pH on the removal efficiency of the dye were examined. Results: The results showed that in the initial dye concentration of 50 mg/L, the optimum amounts of adsorbent and pH were 0.3 g and 7, respectively. Increasing the dye concentration from 20 to 100 mg/L resulted in the reduction of the removal efficiency from 100% to 79% in the contact time of 90 minutes. The results indicated the highest attracting correlation with Langmuir model. The maximum adsorbent capacity obtained from Langmuir model was 25 mg/g. The kinetics of the dye adsorption on the modified zeolite followed pseudo-second-order kinetics model. Calculated thermodynamic parameters showed that Gibbs free energy changes (DGo) at temperatures of 20 and 45°C were -29.41 and -35.20 kJ/mol, respectively. Enthalpy (DHo) and entropy changes were equal to 41.181 kJ/mol and 0.241 J/mol K, respectively. The results showed that the processing was a spontaneous endothermic reaction. The process modeled by artificial neural networks (ANN) showed that the experimental results can be accurately modeled using neural network model. The correlation coefficient found between the experimental and the model results was 0.951. Conclusion: Due to the low cost, high abundance and availability of zeolite, the removal efficiency of this adsorbent can be increased to desirable levels by modifying. Keywords: Zeolite, Adsorption, Kinetics, Thermodynamics, Neural network

A new magnetic bio-sorbent for arsenate removal from the contaminated water: Characterization, isotherms, and kinetics

Background: Arsenic (AS) is a heavy metal pollutant in water that has been known as one of the most important environmental contaminants due to its serious effects on both human health and the environment. This study was conducted to investigate the efficiency of calcined Co/Fe/Al LDH@Fe3O4@ PA as a new magnetic bio-sorbent for AS removal from the polluted water. Methods: At first, magnetic ternary calcined layered double hydroxide (Co/Fe/Al LDH) was synthesized through co-precipitation procedure. The synthesized CLDH was modified with phenylalanine amino acid, named CLDH@Fe3O4@PA. Infrared spectroscopy, X-ray diffraction, transmission, and field emission scanning electron microscopy (FESEM) were used to confirm the synthesis of the sorbent. The removal time, pH, and the sorbent dose were studied and optimized as the effective parameters on the As (V) removal. Results: The XRD, FTIR, TEM, SEM, EDS, and VSM techniques confirmed the properties of the synthesized magnetic bio-sorbent. Based on the optimization study, pH=6, the sorbent concentration of 30 mg, and the removal time of 5 minutes were considered as the optimum conditions with about 91% AS removal. The Langmuir isotherm with higher R2 value was matched well with the obtained results, and values obtained for qm and RL were 167 mg g–1 and 0.976 to 0.993, respectively. The kinetics studies were fitted well with the linear pseudo-first-order model with higher R2 at sorption process. Conclusion: The real samples results confirmed the excellent As (V) sorption capacity of the synthesized magnetic bio-sorbent in comparison with other sorbents. Therefore, CLDH@Fe3O4@PA sorbent is introduced as a new suitable sorbent for removal of As (V) from the polluted water. Keywords: Water pollution, Phenylalanine, Ferrosoferric oxide, Arseni

Peritumoral administration of GPI-anchored TIMP-1 inhibits colon carcinoma growth in Rag-2 gamma chain-deficient mice

Exogenous application of recombinant TIMP-1 protein modified by addition of a glycosylphosphatidylinositol (GPI) anchor allows efficient insertion of the fusion protein into cell membranes. This `cell surface engineering' leads to changes in the proteolytic environment. TIMP-1-GPI shows enhanced as well as novel in vitro biological activities including suppression of proliferation, reduced migration, and inhibition of invasion of the colon carcinoma cell line SW480. Treatment of SW480 tumors implanted in Rag (-/-) common gamma chain (-/-) C57BL/6 mice with peritumorally applied TIMP-1-GPI, control rhTIMP-1 protein, or vehicle shows that TIMP-1-GPI leads to a significant reduction in tumor growth

Recombinant GPI-anchored TIMP-1 stimulates growth and migration of peritoneal mesothelial cells.

Mesothelial cells are critical in the pathogenesis of post-surgical intraabdominal adhesions as well as in the deterioration of the peritoneal membrane associated with long-term peritoneal dialysis. Mesothelial denudation is a pathophysiolocigally important finding in these processes. Matrix metalloproteinase (MMP) biology underlies aspects of mesothelial homeostasis as well as wound repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) moderate MMP activity. METHODS AND FINDING: By modifying human TIMP-1 through the addition of a glycosylphosphatidylinositol (GPI) anchor, a recombinant protein was generated that efficiently focuses TIMP-1 on the cell surface. Treatment of primary mesothelial cells with TIMP-1-GPI facilitates their mobilization and migration leading to a dramatic increase in the rate of wound experimental closure. Mesothelial cells treated with TIMP-1-GPI showed a dose dependent increase in cell proliferation, reduced secretion of MMP-2, MMP-9, TNF-α and urokinase-type plasminogen activator (uPA), but increased tissue plasminogen activator (t-PA). Treatment resulted in reduced expression and processing of latent TGF-β1. TIMP-1-GPI stimulated rapid and efficient in vitro wound closure. The agent enhanced mesothelial cell proliferation and migration and was bioactive in the nanogram range. The application of TIMP-1-GPI may represent a new approach for limiting or repairing damaged mesothelium

Effect of cytomix and sedative drugs on mitochondrial electron transport chain of cultured primary human astrocytes

Septic shock is a major cause of death among patients in intensive care units worldwide. Despite the recent developments and progression in medical research, sepsis remains a challenge. Multiple‐organ failure including brain dysfunction (septic encephalopathy) is the predominant cause of death in septic patients. Elevations of cytokine concentrations in the brain have been described in both experimental and clinical studies. Furthermore, mitochondrial dysfunction has also been described in septic encephalopathy. Since also sedative drugs interfere with brain function, they may contribute to septic encephalopathy

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

AbstractMitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y. lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y. lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron–sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems