Fabrication and Characterization of Cell Membrane

Cell membrane plays critical cellular functions in both physiological and pathological pathways and thus is important target for both basic and applied biomedical research. The domain structure features of cell membrane strongly affect the functions of membrane embedded biomolecules such as proteins and carbohydrates. However, understanding the structural aspects of membrane effects on the embedded biomolecule’s function have not been able easily to do due to limited approaches available. We have engaged in fabrication of cell membrane mimetic systems for functional analysis of membrane proteins and cell surface carbohydrates [1,2]. Thrombomodulin (TM), an endothelial integral membrane protein, plays central roles in haemostatic balance by serving as a cofactor for thrombin-mediated protein C (PC) activation (antithrombotic) [3]. The structure of TM and its structural domains necessary for PC activation has been clarified and reactions occur on endothelial cell membrane surfaces [4]. Therefore, cell membrane may be involved in the protein C activation process. However, it is not conducted so far. In this research, we fabricate cell membrane mimetic systems containing TM and investigate the physiological significance of the lipid membrane on TM-enhanced PC activation mechanism. The proposed research will provide important information to understand the relative factors involved in PC activation and would offer opportunities to manipulate thrombotic disorders (antithrombotic versus prothrombotic) related to cardiovascular diseases. In addition, the cell membrane mimetic system can be used for examining binding interactions of other cell surface biomolecules such as carbohydrates and can be applied for drug screening applications as well.https://engagedscholarship.csuohio.edu/u_poster_2014/1026/thumbnail.jp

Glyco-modification of Protein with O-cyanate Chain-end Functionalized Glycopolymer via Isourea Bond Formation

Glycoengineering aimed at addition of carbohydrates to proteins is an attractive approach to alter pharmacokinetic properties of proteins such as enhancing stability and prolonging the duration of action. We report a novel protein glyco-modification of BSA and recombinant thrombomodulin with O-cyanate chain-end functionalized glycopolymer via isourea bond formation. The protein glycoconjugates were confirmed by SDS-PAGE, western blot, and MALDI-TOF Mass Spectrometry. Protein C activation activity of the glyco-modified recombinant thrombomodulin was confirmed, proving no interference to activity from the glycopolymer modification. The isourea bond formation under mild conditions was demonstrated as an alternative method for protein modification with polymers

Investigation of Cofactor Activities of Endothelial Microparticle- Thrombomodulin with Liposomal Surrogate

Thrombomodulin (TM) is a type I transmembrane glycoprotein mainly expressed on the endothelial cells, where it binds thrombin to form the thrombin-TM complex that can activate protein C and thrombin-activable fibrinolysis inhibitor (TAFI) and induce anticoagulant and anti-fibrinolytic reactions, respec-tively. Cell activation and injury often sheds microparticles that contain membrane TM, which circulate in biofluids like blood. However, the biological function of circulating microparticle-TM is still unknown even though it has been recognized as a biomarker of endothelial cell injury and damage. In comparison with cell membrane, different phospholipids are exposed on the microparticle surface due to cell membrane flip-flop upon cell activation and injury. Liposomes can be used as a microparticle mimetics. In this report, we prepared TM-containing liposomes with different phospholipids as surrogates of endothelial microparticle-TM and investigated their cofactor activities. We found that liposomal TM with phosphatidylethanolamine (PtEtn) showed increased protein C activation but decreased TAFI activation in comparison to liposomal TM with phosphatidylcholine (PtCho). In addition, we investigated whether protein C and TAFI compete for the thrombin/TM complex on the liposomes. We found that protein C and TAFI did not compete for the thrombin/TM complex on the liposomes with PtCho alone and with low concentration (5%) of PtEtn and phosphatidylserine (PtSer), but competed each other on the liposomes with higher concentration (10%) of PtEtn and PtSer. These results indicate that membrane lipids affect protein C and TAFI activation and microparticle-TM may have different cofactor activities in comparison to cell membrane TM.(c) 2023 The Author(s)

The connection analysis between the dilution of the deposited Fe-Cr-V-Mo-C layer by the basic metal and the parameters of its microstructure

In this work, the structure of the Fe-Cr-V-Mo-C coatings received by plasma transferred arc cladding was investigated. Coatings were deposited on plates with a thickness of 10 mm and made from constructional steel (steel 20). The correlation analysis of relationships between dilution of the deposited layers by the basic metal and the parameters of their microstructure was carried out. The parameters were as follows: volume fraction, a size, a shape factor, the distance between particles, the number of particles of vanadium carbide, volume fraction of the eutectic on the basis of carbide M[7]C[3] and the distances between its colonies, as well as the volume fraction of the [alpha]-phase in the alloy matrix

Lietuvos darbo žmonių veikla, kuriant mokyklų materialinę bazę socializmo statybos laikotarpiu (1944-1952 m.)

When Soviet Lithuania was liberated from the German invaders, together with the restoration and development of the national economy popular education was in the centre of attention of the Lithuanian Communist party and the Soviet Government. A successful development of the national economy and a constant support of our sister republics made it possible to increase the assignations to popular education every year. The assigned sums let us successfully put into life the undertaken task by our Party - compulsory seven-year education - and create conditions for passing on to universal secondary education. The best necessary conditions were made for a normal educational work at schools. They were provided with premises and school supplies. Teachers, workers ant youth showed a great initiative and patriotism. Thanks to them in our republic a new way of building schools began spreading. Hundreds of schools for tens of thousands of working places were reconstructed and built. Sister republics gave a great help in restoring schools and developing their material basis.Kai Sovietinė Lietuva buvo išvaduota iš Vokietijos okupacijos, kartu su nacionalinės ekonomikos atstatymu ir vystymu, Lietuvos Komunistų partijos ir Sovietų valdžios dėmesys buvo skiriamas ir visuotiniam švietimui. Sėkmingas nacionalinės ekonomikos augimas ir pastovi šalių seserų parama suteikė galimybę kasmet didinti paskyrimus visuotiniam švietimui. Paskirtos sumos leido sėkmingai įgyvendinti Partijos vykdomą užduotį – privalomą septynerių metų išsilavinimą – ir sukurti sąlygas tęsti mokslus ir įgytį bendrą vidurinį išsilavinimą. Buvo suteiktos reikiamos sąlygos darbui mokykloje: aprūpinta patalpomis ir mokyklos reikmenimis. Mokytojai, darbuotojai ir jaunimas pademonstravo didelį iniciatyvumą ir patriotizmą. Jų dėka mūsų valstybėje ėmė plisti naujas mokyklų kūrimo būdas, buvo rekonstruota šimtai mokyklų su dešimtimis tūkstančių darbo vietų. Seserinės šalys suteikė didžiulę pagalbą atstatant mokyklas ir kuriant jų materialinę bazę

Fabrication and Characterization of Cell Membrane

Cell membrane plays critical cellular functions in both physiological and pathological pathways and thus is important target for both basic and applied biomedical research. The domain structure features of cell membrane strongly affect the functions of membrane embedded biomolecules such as proteins and carbohydrates. However, understanding the structural aspects of membrane effects on the embedded biomolecule’s function have not been able easily to do due to limited approaches available. We have engaged in fabrication of cell membrane mimetic systems for functional analysis of membrane proteins and cell surface carbohydrates [1,2]. Thrombomodulin (TM), an endothelial integral membrane protein, plays central roles in haemostatic balance by serving as a cofactor for thrombin-mediated protein C (PC) activation (antithrombotic) [3]. The structure of TM and its structural domains necessary for PC activation has been clarified and reactions occur on endothelial cell membrane surfaces [4]. Therefore, cell membrane may be involved in the protein C activation process. However, it is not conducted so far. In this research, we fabricate cell membrane mimetic systems containing TM and investigate the physiological significance of the lipid membrane on TM-enhanced PC activation mechanism. The proposed research will provide important information to understand the relative factors involved in PC activation and would offer opportunities to manipulate thrombotic disorders (antithrombotic versus prothrombotic) related to cardiovascular diseases. In addition, the cell membrane mimetic system can be used for examining binding interactions of other cell surface biomolecules such as carbohydrates and can be applied for drug screening applications as well.https://engagedscholarship.csuohio.edu/u_poster_2014/1026/thumbnail.jp

Glyco-modification of Protein with O-cyanate Chain-end Functionalized Glycopolymer via Isourea Bond Formation

Glycoengineering aimed at addition of carbohydrates to proteins is an attractive approach to alter pharmacokinetic properties of proteins such as enhancing stability and prolonging the duration of action. We report a novel protein glyco-modification of BSA and recombinant thrombomodulin with O-cyanate chain-end functionalized glycopolymer via isourea bond formation. The protein glycoconjugates were confirmed by SDS-PAGE, western blot, and MALDI-TOF Mass Spectrometry. Protein C activation activity of the glyco-modified recombinant thrombomodulin was confirmed, proving no interference to activity from the glycopolymer modification. The isourea bond formation under mild conditions was demonstrated as an alternative method for protein modification with polymers

Azide-Reactive Liposome for Chemoselective and Biocompatible Liposomal Surface Functionalization and Glyco-Liposomal Microarray Fabrication

Chemically selective liposomal surface functionalization and liposomal microarray fabrication using azide-reactive liposomes are described. First, liposome carrying PEG-triphenylphosphine was prepared for Staudinger ligation with azide-containing biotin, which was conducted in PBS buffer (pH 7.4) at room temperature without a catalyst. Then, immobilization and microarray fabrication of the biotinylated liposome onto a streptavidin-modified glass slide via the specific streptavidin/biotin interaction were investigated by comparing with directly formed biotin–liposome, which was prepared by the conventional liposome formulation of lipid–biotin with all other lipid components. Next, the covalent microarray fabrication of liposome carrying triphenylphosphine onto an azide-modified glass slide and its further glyco-modification with azide-containing carbohydrate were demonstrated for glyco-liposomal microarray fabrication via Staudinger ligation. Fluorescence imaging confirmed the successful immobilization and protein binding of the intact immobilized liposomes and arrayed glyco-liposomes. The azide-reactive liposome provides a facile strategy for membrane-mimetic glyco-array fabrication, which may find important biological and biomedical applications such as studying carbohydrate–protein interactions and toxin and antibody screening

Chemoselectively Surface Funtionalizable Tethered Bilayer Lipid Membrane for Versatile Membrane Mimetic Systems Fabrication

A chemoselectively surface functionalizable tethered bilayer lipid membrane (tBLM) was developed via liposome immobilization, rupture and fusion processes. Briefly, introduction of triphenylphosphine (TP)-PEG-lipid into a liposome allows it to immobilize onto an azide surface through amide bond formation via Staudinger ligation. Subsequent rupture of the immobilized liposome followed by a second liposome fusion leads to the tBLM formation, which contains TP for further chemically selective modifications on its surface. The membrane fluidity and continuity of the tBLM were confirmed by confocal fluorescence microscopy. The tBLM was covalently functionalized with biomolecules such as azide-containing glycan and biotin in chemically selective fashion and under biocompatible condition, and thus provides a straightforward approach for multifunctional membrane mimetic system fabrication. In addition, the tBLM with incorporated transmembrane protein was demonstrated with an endothelial membrane protein thrombomodulin and its protein C activity was confirmed. The tBLM is very versatile as it can be adapted easily to different types of supporter for a variety of biological and biomedical research areas and applications