Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.

Polyvanadate-stimulated NADH oxidation by plasma membranes-The need for a mixture of deca and meta forms of vanadate

Polyvanadate solutions obtained by extracting vanadium pentoxide with dilute alkali over a period of several hours contained increasing amounts of decavanadate as characterized by NMR and ir spectra. Those solutions having a metavanadate:decavanadate ratio in the range of 1-5 showed maximum stimulation of NADH oxidation by rat liver plasma membranes. Reduction of decavanadate, but not metavanadate, was obtained only in the presence of the plasma membrane enzyme system. High simulation of activity of NADH oxidation was obtained with a mixture of the two forms of vanadate and this further increased on lowering the pH. Addition of increasing concentrations of decavanadate to metavanadate and vice versa increased the stimulatory activity, reaching a maximum when the metavanadate:decavanadate ratio was in the range of 1-5. Increased stimulatory activity can also be obtained by reaching these ratios by conversion of decavanadate to metavanadate by alkaline phosphate degradation, and of metavanadate to decavanadate by acidification. These studies show for the first time that both deca and meta forms of vanadate present in polyvanadate solutions are needed for maximum activity of NADH oxidation.

Thin nanoporous metal-insulator-metal membranes

Insulating nanoporous materials are promising platforms for soft-ionizing membranes; however, improvement in fabrication processes and the quality and high breakdown resistance of the thin insulator layers are needed for high integration and performance. Here, scalable fabrication of highly porous, thin, silicon dioxide membranes with controlled thickness is demonstrated using plasma-enhanced chemical-vapor-deposition. The fabricated membranes exhibit good insulating properties with a breakdown voltage of 1 × 107 V/cm. Our calculations suggest that the average electric field inside a nanopore of the membranes can be as high as 1 × 106 V/cm; sufficient for ionization of wide range of molecules. These metal–insulator–metal nanoporous arrays are promising for applications such soft ionizing membranes for mass spectroscopy.

Novel organic-inorganic composite polymer-electrolyte membranes for DMFCs

Organic-inorganic composite membranes comprising Nation with inorganic materials such as silica, mesoporous zirconium phosphate (MZP) and mesoporous titanium phosphate (MTP) are fabricated and evaluated as proton-exchange-membrane electrolytes for direct methanol fuel cells (DMFCs). For Nation-silica composite membrane, silica is impregnated into Nation matrix as a sol by a novel water hydrolysis process precluding the external use of an acid. Instead, the acidic nature of Nation facilitates in situ polymerization reaction with Nation leading to a uniform composite membrane. The rapid hydrolysis and polymerization reaction while preparing zirconia and titania sols leads to uncontrolled thickness and volume reduction in the composite membranes, and hence is not conducive for casting membranes. Nafion-MZP and Nafion-MTP composite membranes are prepared by mixing pre-formed porous MZP and MTP with Nation matrix. MZP and MTP are synthesised by co-assembly of a tri-block co-polymer, namely pluronic-F127, as a structure-directing agent, and a mixture of zirconium butoxide/titanium isopropoxide and phosphorous trichloride as inorganic precursors. Methanol release kinetics is studied by volume-localized NMR spectroscopy (employing ``point resolved spectroscopy'', PRESS), the results clearly demonstrating that the incorporation of inorganic fillers in Nation retards the methanol release kinetics under osmotic drag. Appreciable proton conductivity with reduced methanol permeability across the composite membranes leads to improved performance of DMFCs in relation to commercially available Nafion-117 membrane.

Cereal alkylresorcinols as dietary biomarkers-absorption and occurrence in biological membranes

Several studies link the consumption of whole-grain products to a lowered risk of chronic diseases, such as certain types of cancer, type II diabetes, and cardiovascular diseases. However, the final conclusions of the exact protective mechanisms remain unclear, partly due to a lack of a suitable biomarker for the whole-grain cereals intake. Alkylresorcinols (AR) are phenolic lipids abundant in the outer parts of wheat and rye grains usually with homologues of C15:0- C25:0 alkyl chains, and are suggested to function as whole-grain biomarkers. Mammalian lignan enterolactone has also previously been studied as a potential whole-grain biomarker. In the present work a quantified gas chromatography-mass spectrometry method for the analysis of AR in plasma, erythrocytes, and lipoproteins was developed. The method was used to determine human and pig plasma AR concentrations after the intake of whole-grain wheat and rye products compared to low-fibre wheat bread diets to assess the usability of AR as biomarkers of whole-grain intake. AR plasma concentrations were compared to serum ENL concentrations. AR absorption and elimination kinetics were investigated in a pig model. AR occurrence in human erythrocyte membranes and plasma lipoproteins were determined, and the distribution of AR in blood was evaluated. Plasma AR seem to be absorbed via the lymphatic system from the small intestine, like many other lipophilic compounds. Their apparent elimination half-life is relatively short and is similar to that of tocopherols, which have a similar chemical structure. Plasma AR concentrations increased significantly after a one- to eight-week intake of whole-grain wheat and further on with whole-grain rye bread. The concentrations were also higher after habitual Finnish diet compared to diet with low-fibre bread. Inter-individual variation after a one-week intake of the same amount of bread was high, but the mean plasma AR concentrations increased with increasing AR intake. AR are incorporated into erythrocyte membranes and plasma lipoproteins, and VLDL and HDL were the main AR carriers in human plasma. Based on these studies, plasma AR could function as specific biomarkers of dietary whole-grain products. AR are exclusively found in whole-grains and are more suitable as specific biomarkers of whole-grain intake than previously investigated mammalian lignan enterolactone, that is formed from several plants other than cereals in the diet. Plasma AR C17:0/C21:0 -ratio could distinguish whether whole-grain products in the diet are mainly wheat or rye. AR could be used in epidemiological studies to determine whole-grain intake and to better assess the role of whole-grains in disease prevention.

Mechanical response of the tympanal membranes of the tree cricket Oecanthus henryi

Crickets have two tympanal membranes on the tibiae of each foreleg. Among several field cricket species of the genus Gryllus (Gryllinae), the posterior tympanal membrane (PTM) is significantly larger than the anterior membrane (ATM). Laser Doppler vibrometric measurements have shown that the smaller ATM does not respond as much as the PTM to sound. Hence the PTM has been suggested to be the principal tympanal acoustic input to the auditory organ. In tree crickets (Oecanthinae), the ATM is slightly larger than the PTM. Both membranes are structurally complex, presenting a series of transverse folds on their surface, which are more pronounced on the ATM than on the PTM. The mechanical response of both membranes to acoustic stimulation was investigated using microscanning laser Doppler vibrometry. Only a small portion of the membrane surface deflects in response to sound. Both membranes exhibit similar frequency responses, and move out of phase with each other, producing compressions and rarefactions of the tracheal volume backing the tympanum. Therefore, unlike field crickets, tree crickets may have four instead of two functional tympanal membranes. This is interesting in the context of the outstanding question of the role of spiracular inputs in the auditory system of tree crickets.

Nafion and modified-Nafion membranes for polymer electrolyte fuel cells: An overview

Polymer electrolyte fuel cells (PEFCs) employ membrane electrolytes for proton transport during the cell reaction. The membrane forms a key component of the PEFC and its performance is controlled by several physical parameters, viz. water up-take, ion-exchange capacity, proton conductivity and humidity. The article presents an overview on Nafion membranes highlighting their merits and demerits with efforts on modified-Nafion membranes.

Improved procedures for static and dynamic analyses of wrinkled membranes

An analysis of large deformations of flexible membrane structures within the tension field theory is considered. A modification-of the finite element procedure by Roddeman et al. (Roddeman, D. G., Drukker J., Oomens, C. W J., Janssen, J. D., 1987, ASME J. Appl. Mech. 54, pp. 884-892) is proposed to study the wrinkling behavior of a membrane element. The state of stress in the element is determined through a modified deformation gradient corresponding to a fictive nonwrinkled surface. The new model uses a continuously modified deformation gradient to capture the location orientation of wrinkles more precisely. It is argued that the fictive nonwrinkled surface may be looked upon as an everywhere-taut surface in the limit as the minor (tensile) principal stresses over the wrinkled portions go to zero. Accordingly, the modified deformation gradient is thought of as the limit of a sequence of everywhere-differentiable tensors. Under dynamic excitations, the governing equations are weakly projected to arrive at a system of nonlinear ordinary differential equations that is solved using different integration schemes. It is concluded that, implicit integrators work much better than explicit ones in the present context.

Thermotropic and hydration studies of membranes formed from gemini pseudoglyceryl lipids possessing polymethylene spacers

Membrane formation from gemini pseudoglyceryl lipids bearing n-C14H29 and n-C16H33 chains has been reported. These lipid aggregates have been characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), high sensitivity differential scanning calorimetry (DSC), and Paldan fluorescence studies. The length of the spacer between the cationic ammonium headgroups has been varied from -(CH2)(3)- (propandiyl) to -(CH2)(12)- (dodecandiyl) in these lipids. All gemini lipids were found to generate stable suspensions in aqueous media. Electron microscopic studies revealed the smaller size of the gemini lipid aggregates as compared to their monomeric lipid counterparts. DLS measurements showed that the gemini lipid suspensions with a -(CH2)(8)- spacer length were bigger in size than that of other analogues. DSC studies suggest the unusual behavior of the gemini lipids bearing -(CH2)3- propanediyl spacer based lipids. These observations were consistent irrespective of the hydrocarbon chain lengths of the lipids. Paldan fluorescence based hydration studies showed that the hexadecyl chain based gemini lipid aggregates bearing a -(CH2)(12)- spacer were the most hydrated in their gel states among all the gemini lipid series investigated herein.

Understanding Membranes through the Molecular Design of Lipids

Lipids are amphiphilic molecules that are composed of hydrophilic and hydrophobic regions. A typical membranous aggregate (vesicles, water-filled lipid nanospheres) is formed upon the self-organization of lipids in water from a diverse collection of amphiphiles producing a dynamic supramolecular structure that shows phase behavior and ordering as required for specific biological functions. The determination of various physical properties of lipid aggregates is the key to determining structure-function relationships. Over the years, we have designed and synthesized a wide variety of lipid molecular systems for the investigation of their membrane-forming properties and have used them for purposes such as gene delivery and enzyme activation. In this feature article, we focus on our work on various types of lipids including ion-paired amphiphiles, cholesterol-based lipids, aromatic lipids, macrocyclic lipids containing disulfide tethers; cationic dimeric lipids, and so forth. The emphasis is oil experimental design and bottom-line conclusions.

Export of proteins across membranes: The helix reversion hypothesis

A model is presented which explains the biological role of the leader peptide in protein export. Along the lines of this model, the conformational changes of a protein with environment serves as a general mechanism for translocation. The leader peptide in the cytoplasm takes a hairpin like conformation which reverts to an extended helix upon integration into the membrane. The essential features of this model are in accord with recent results of protein export.